Targeted nitroxide agents

ABSTRACT

Provided herein are compositions and related methods useful for free radical scavenging, with particular selectivity for mitochondria. The compounds comprise a nitroxide-containing group attached to a mitochondria-targeting group. The compounds can be cross-linked into dimers without loss of activity. Also provided herein are methods, for preventing, mitigating and treating damage caused by radiation. The method comprises delivering a compound, as described herein, to a patient in an amount and dosage regimen effective to prevent, mitigate or treat damage caused by radiation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to UnitedStates Provisional Patent Application Nos. 61/081,585, filed on Jul. 17,2008; 61/158,569, filed on Mar. 9, 2009; and 61/178,570, filed on May15, 2009, each of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERAL FUNDING

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.NIAID U19 AI068021, awarded by the National Institutes of Health.

Provided herein are novel compounds and compositions of mattercomprising a nitroxide group-containing cargo (or “nitroxide containinggroup”) and a mitochondria-targeting group (or “targeting group”). Thetargeting group is believed, without any intent to be bound, to have theability to selectively deliver the composition to mitochondrial,delivering the antioxidant and free-radical scavenging activity of thenitroxide group to cells, including but not limited to an enrichment inmitochondria. These compounds are useful, generally, for theiranti-oxidant and free-radical scavenging capacity, and morespecifically, for example and without limitation, for theirradioprotective abilities and prevention as well as mitigation ofdegenerative diseases.

Oxidation stress in cells typically manifests itself by way ofgenerating reactive oxygen species (“ROS”) and reactive nitrogen species(“RNS”). Specifically, the cellular respiration pathway generates ROSand RNS within the mitochondrial membrane of the cell, see Kelso et al.,Selective Targeting of a Redox-active Ubiquinone to Mitochondria withinCells: Antioxidant and Antiapoptotic Properties, J Biol Chem. 276:4588(2001). Reactive oxygen species include free radicals, reactive anionscontaining oxygen atoms, and molecules containing oxygen atoms that caneither produce free radicals or are chemically activated by them.Specific examples include superoxide anion, hydroxyl radical, andhydroperoxides. In many disease states, the normal response to ROS andRNS generation is impaired.

Naturally occurring enzymes, such as superoxide dismutase (“SOD”) andcatalase salvage ROS and RNS radicals to allow normal metabolic activityto occur. Significant deviations from cell homeostasis, such ashemorrhagic shock, lead to an oxidative stress state, thereby causing“electron leakage” from the mitochondrial membrane, this “electronleakage” produces an excess amount of ROS for which the cell's naturalantioxidants cannot compensate. Specifically, SOD cannot accommodate theexcess production of ROS associated with hemorrhagic shock whichultimately leads to premature mitochondria dysfunction and cell deathvia apoptosis, see Kentner et al., Early Antioxidant Therapy with TEMPOLduring Hemorrhagic Shock Increases Survival in Rats, J Trauma Inj InfectCrit Care., 968 (2002).

Cardiolipin (“CL”) is an anionic phospholipid exclusively found in theinner mitochondrial membrane of eukaryotic cells, sec Iverson, S. L. andS. Orrenius, The cardiolipincytochrome c interaction and themitochondria) regulation of apoptosis, Arch Biochem. 423:37-46 (2003).Under normal conditions, the pro-apoptotic protein cytochrome c isanchored to the mitochondrial inner membrane by binding with CL, seeTuominen, E. K. J., et al. Phospholipid cytochrome c interaction:evidence for the extended lipid anchorage, J Biol Chem., 277:8822-8826(2002). The acyl moieties of CL are susceptible to peroxidation byreactive oxygen species. When ROS are generated within mitochondria inexcess quantities, cytochrome C bound to CL can function as an oxidaseand induces extensive peroxidation of CL in the mitochondrial membrane,see Kagan, V. E. et al., Cytochrome c acts as a cardiolipin oxygenaserequired, for release of proapoptotic, factors, Nat Chem Biol. 1:223-232(2005); also Kagan, V. E. et al., Oxidative lipidomics of apoptosis:redox catalytic interactions of cytochrome c with cardiolipin andphosphatidylserine, Free Rad Biol Med. 37:1963-1985 (2005).

The peroxidation of the CL weakens the binding between the CL andcytochrome C, see Shidoji, Y. et al., Loss of molecular interactionbetween cytochrome C and cardiolipin due to lipid peroxidation. BiochemBiophys Res Comm. 264:343-347 (1999). This leads to the release of thecytochrome C into the mitochondrial intermembrane space, inducingapoptotic cell death. Further, the peroxidation of CL has the effect ofopening the mitochondrial permeability transition pore (“MPTP”), seeDolder, M. et al., Mitochondria creatine kinase in contact sites:Interaction with porin and adenine nucleotide translocase, role inpermeability transition and sensitivity to oxidative damage, Biol SignRecept, 10:93-111 (2001); also Imai, H. et al., Protection frominactivation of the adenine nucleotide translocator duringhypoglycaemia-induced apoptosis by mitochondria/phospholipidhydroperoxide glutathione peroxidase, Biochem J., 371:799-809 (2003).Accordingly, the mitochondrial membrane swells and releases thecytochrome C into the cytosol. Excess cytochrome C in the cytosol leadsto cellular apoptosis, see Iverson, S. L. et al. Thecardiolipin-cytochrome c interaction and the mitochondria regulation ofapoptosis, Arch Biochem Biophys. 423:37-46 (2003).

Moreover, mitochondrial dysfunction and cell death may ultimately leadto multiple organ failure despite resuscitative efforts or supplementaloxygen supply, see Cairns, C., Rude Unhinging of the Machinery of Life:Metabolic approaches to hemorrhagic Shock, Curr Crit Care., 7:437(2001). Accordingly, there is a need in the art for an antioxidant whichscavenges the ROS, thereby reducing oxidative stress. Reduction ofoxidative stress delays, even inhibits, physiological conditions thatotherwise might occur, such as hypoxia.

Also, there is also a need to improve the permeability of antioxidants'penetration of the cellular membrane. One of the limitations of SOD isthat it cannot easily penetrate the cell membrane. However, nitroxideradicals, such as TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) and itsderivatives, have been shown to penetrate the cell membrane better thanSOD. Further, nitroxide radicals like, for example and withoutlimitation, TEMPO prevent the formation of ROS, particularly superoxide,due to their reduction by the mitochondrial electron transport chain tohydroxyl amine radical scavengers, see Wipf. P. et al., Mitochondriatargeting of selective electron scavengers: synthesis and biologicalanalysis of hemigramicidin-TEMPO conjugates, J Am Chem Soc.127:12460-12461. Accordingly, selective delivery of TEMPO derivativesmay lead to a therapeutically beneficial reduction of ROS and may delayor inhibit cell death due to the reduction of oxidative stress on thecell.

Selective delivery may be accomplished by way of a number of differentpathways—e.g., by a biological or chemical moiety having a specifictargeting sequence for penetration of the cell membrane, ultimatelybeing taken up by the mitochondrial membrane. Selective delivery of anitroxide SOD mimic into the mitochondrial membrane has provendifficult. Accordingly, there is a need in the art for effective andselective delivery of antioxidants that specifically target themitochondria and its membranes as well as inter-membrane space to helpreduce the ROS and RNS species. The antioxidants also help preventcellular and mitochondria apoptotic activity which often results due toincreased ROS species, see Kelso et al., Selective Targeting of aRedox-active Ubiquinone to Mitochondria within Cells: Antioxidant andAntiapoptotic Properties, J Biol Chem., 276: 4588 (2001). Examples ofmitochondria-targeting antioxidants are described in United StatesPatent Publication Nos. 20070161573 and 20070161544.

There remains a very real need for a composition and associated methodsfor delivering cargo of various types to mitochondria. In oneembodiment, a composition comprising membrane active peptidyl fragmentshaving a high affinity with the mitochondria linked to cargo isprovided. The cargo may be selected from a large group of candidates.There is a need for compositions and methods for effectively treating acondition that is caused by excessive mitochondria production of ROS andRNS in the mitochondrial membrane. There also is a need for compoundsthat can protect cells and tissues of animals against radiation damage.

Radiation Exposure

The biologic consequences of exposure to ionizing radiation (IR) includegenomic instability and cell death (Little J B, Nagasawa H, Pfenning T,et al. Radiation-induced genomic instability: Delayed mutagenic andcytogenetic effects of X rays and alpha particles. Radial Res 1997;148:299-307). It is assumed that radiolytically generated radicals arethe primary cause of damage from 1R. Direct radiolysis of water and thesecondary reactive intermediates with a short lifetime (10⁻¹⁰-10⁶seconds) mediate the chemical reactions that trigger the damage ofcellular macromolecules, including DNA and proteins, as well asphospholipids in membranes (Mitchell J B, Russo A, Kuppusamy P, et al.Radiation, radicals, and images. Ann N Y Acad Sci 2000; 899:28-43). TheDNA is believed to be the primary target for the radical attack,resulting in single and double DNA strand breaks (Bryant P E. Enzymaticrestriction of mammalian cell DNA: Evidence for double-strand breaks aspotentially lethal lesions. Int J Radial Biol 1985; 48:55-60). Tomaintain the genomic integrity, multiple pathways of DNA repair andcell-cycle checkpoint control are activated in response toirradiation-induced DNA damage (Elledge S J. Cell cycle checkpoints:Preventing an identity crisis. Science 1996; 274:1664-1672). Failure ofthese repair and regulatory systems leads to genotoxicity, malignanttransformation, and cell death (Sachs R K, Chen A M, Brenner D J.Proximity effects in the production of chromosome aberrations byionizing radiation. Int J Radiat Biol 1997; 71:1-19).

One of the major mechanisms of IR-induced cell death is apoptosis, mostcommonly realized through a mitochondria-dependent intrinsic pathway(Newton K, Strasser A. Ionizing radiation and chemotherapeutic drugsinduce apoptosis in lymphocytes in the absence of Fas or FADD/MORT1signaling. Implications for cancer therapy. J Exp Med 2000;191:195-200). The latter includes permeabilization of mitochondriafollowed by the release of cytochrome (cyt) c and other proapoptoticfactors (Smac/Diablo [second mitochondrial-derived activator ofcaspase/direct inhibitor of apoptosis-binding protein with low pI],EndoG [endonuclease G], Omi/HtrA2, and AIF [apoplosisinducing factor])into the cytosol as the key events in the execution of the deathprogram. The released cyt c facilitates the formation of apoptosomes byinteracting with apoptotic protease activating factor 1 (Apaf-1) andthen recruits and activates procaspase-9 and triggers the proteolyticcascade that ultimately leads to cell disintegration. Release ofproapoptotic factors and caspase activation designate the commencementof irreversible stages of apoptosis. Therefore, significant drugdiscovery efforts were directed toward the prevention of these events,particularly of the mitochondrial injury representing an important pointof no return (Szewczyk A, Wojtczak L. Mitochondria as a pharmacologicaltarget. Pharmacol Rev 2002; 54:101-127). However, the exact mechanismsof cyt c release from mitochondria are still poorly understood. It waspostulated that generation of reactive oxygen species (ROS), likely bymeans of disrupted electron transport, has a crucial role in promotingcyt c release from mitochondria (Kowaltowski A J, Castilho R F, VercesiA E. Opening of the mitochondrial permeability transition pore byuncoupling or inorganic phosphate in the presence of Ca2+ is dependenton mitochondrial-generated reactive oxygen species. FEBS Lett 1996;378:150-152). Notably, ROS can induce mitochondria membranepermeabilization both in vitro and in vivo, and the mitochondrialmembrane transition pore was shown to be redox sensitive (Kroemer G,Reed J C. Mitochondrial control of cell death. Nat Med 2000; 6:513-519).

Conversely, antioxidants and reductants, overexpression of manganesesuperoxide dismutase (MnSOD) (Wong G H, Elwell J H, Oberley L W, et al.Manganous superoxide dismutase is essential for cellular resistance tocytotoxicity of tumor necrosis factor. Cell 1989; 58:923-931), andthioredoxin (Iwata S, Hori T, Sato N, et al. Adult T cell leukemia(ATL)-derived factor/human thioredoxin prevents apoptosis of lymphoidcells induced by L-cystine and glutathione depletion: Possibleinvolvement of thiol-mediated redox regulation in apoptosis caused bypro-oxidant stale. J Immunol 1997; 158:3108-3117) can delay or inhibitapoptosis. Previous studies showed that early in apoptosis, amitochondria-specific phospholipid-cardiolipin (CL) translocated fromthe inner to the outer mitochondrial membrane and activated cyt c into aCL-specific peroxidase (Fernandez M G, Troiano L, Moretti L, et al.Early changes in intramitochondrial cardiolipin distribution duringapoptosis. Cell Growth Differ 2002; 13:449-455 and Kagan V E, Tyurin VA, Jiang J, et al. Cytochrome c acts as a cardiolipin oxygenase requiredfor release of proapoptotic factors. Nat Chem Biol 2005; 1:223-232). Theactivated cyt c further catalyzed the oxidation of CL by usingmitochondrially generated ROS (Kagan V E, Tyurin V A, Jiang J, et al.Cytochrome c acts as a cardiolipin oxygenase required for release ofproapoptotic factors. Nat Chem Biol 2005; 1:223-232). Most importantly,oxidized CL is an important contributor to the release of cyt c frommitochondria (Kagan V E, Tyurin V A, Jiang J, et al. Cytochrome c actsas a cardiolipin oxygenase required for release of proapoptotic factors.Nat Chem Biol 2005; 1:223-232 and Petrosillo G, Casanova G, Matera M, etal. Interaction of peroxidized cardiolipin with rat-heart mitochondrialmembranes: Induction of permeability transition and cytochrome crelease. FEBS Lett 2006; 580:6311-6316), which might be attributed tochanges in microenvironment for the interaction between thisphospholipid and cyt c (Ott M, Robertson J D, Gogvadze V, et al.Cytochrome c release from mitochondria proceeds by a two-step process.Proc Nad Acad Sci USA 2002; 99:1259-1263 and Garrido C, Galluzzi L,Brunet M. et al. Mechanisms of cytochrome c release from mitochondria.Cell Death Differ 2006; 13:1423-1433) and/or participation of oxidizedCL in the formation of mitochondrial permeability transition pones (MTP)in coordination with Bcl-2 family proteins (Bid, Bax/Bak), as well asadenine nucleotide translocator (ANT) and voltage-dependent anionchannel (VDAC) (Petrosillo G, Casanova G, Matera M. et al. Interactionof peroxidized cardiolipin with rat-heart mitochondrial membranes:Induction of permeability transition and cytochrome c release. FEBS Lett2006; 580:6311-6316 and Gonzalvez F, Gottlieb E. Cardiolipin: Settingthe beat of apoptosis. Apoptosis 2007; 12:877-885). In addition to theiressential role in the apoptotic signaling pathway, ROS were alsoimplicated in perpetuation of the bystander effect (Narayanan P K,Goodwin E H, Lehnert B E. Alpha particles initiate biological productionof superoxide anions and hydrogen peroxide in human cells. Cancer Res1997; 57:3963-3971 and Iyer R, Lehnert B E. Factors underlying the cellgrowth-related bystander responses to alpha particles. Cancer Res 2000;60:1290-1298) and genomic instability after irradiation exposure (SpitzD R, Azzam E I, Li J J, et al. Metabolic oxidation/reduction reactionsand cellular responses to ionizing radiation: A unifying concept instress response biology. Cancer Metastasis Rev 2004; 23:311-322; LimoliC L, Giedzinski E, Morgan W F, et al. Persistent oxidative stress inchromosomally unstable cells. Cancer Res 2003; 63:3107-3111; and Kim GJ, Chandrasekaran K, Morgan W F. Mitochondrial dysfunction, persistentlyelevated levels of reactive oxygen species and radiation-induced genomicinstability: A review. Mutagenesis 2006; 21:361-367). Hence, eliminationof intracellular ROS, particularly its major source, mitochondrial ROS,by antioxidants may be an important opportunity for developingradioprotectors and radiomitigators. Protection by antioxidants againstIR has been studied for more than 50 years (Weiss J F, Landauer M R.Radioprotection by antioxidants. Ann N Y Acad Sci 2000; 899:44-60).

One of the major mechanisms of ionizing irradiation induced cell deathis apoptosis, most commonly realized through a mitochondria dependentintrinsic pathway. Oxidation of cardiolipin catalyzed by cytochrome c(“cyt c”), release of cytochrome c and other pro-apoptotic factors intothe cytosol and subsequent caspase activation are the key events in theexecution of the death program designating the commencement ofirreversible stages of apoptosis.

In Belikova, N A, et al., (Cardiolipin-Specific Peroxidase Reactions ofCytochrome C in Mitochondria During Irradiation-Induced Apoptosis, Int.J. Radiation Oncology Biol. Phys., Vol. 69, No. 1, pp. 176-186, 2007), asmall interfering RNA (siRNA) approach was used to engineer HeLa cellswith lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treatedby γ-irradiation (in doses of 5-40 Gy). Lipid oxidation wascharacterized by electrospray ionization mass spectrometry analysis andfluorescence highperformance liquid chromatography-based Amplex Redassay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) wasdetected by Western blotting. Apoptosis was revealed by caspase-3/7activation and phosphatidylserine externalization. They showed thatirradiation caused selective accumulation of hydroperoxides incardiolipin (CL) but not in other phospholipids. HeLa 1.2 cellsresponded by a lower irradiation-induced accumulation of CL oxidationproducts than parental HeLa cells. Proportionally decreased release of aproapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cellsafter irradiation. Caspase-3/7 activation and phosphatidylserineexternalization were proportional to the cyt c content in cells. Theyconcluded that cytochrome c is an important catalyst of CL peroxidation,critical to the execution of the apoptotic program. This new role of cytc in irradiation-induced apoptosis is essential for the development ofnew radioprotectors and radiosensitizers.

Significant drug discovery efforts have been directed towards preventionof these events, particularly of the mitochondrial injury thatrepresents an important point of no return. Although the exactmechanisms are still not well understood, generation of reactive oxygenspecies (ROS) and oxidation of cardiolipin by the peroxidase function ofcytochrome c/cardiolipin complexes are believed to play a critical rolein promoting cytochrome c release from mitochondria. ROS—superoxideradicals dismutating to H₂O₂—feed the peroxidase cycle and facilitateaccumulation of oxidized cardiolipin. Hence, elimination ofintracellular ROS, particularly its major source, mitochondrial ROS, byelectron and radical scavengers is a promising opportunity fordeveloping radioprotectors and radiomitigators. Significant research hasbeen conducted in the field of radiation protection to use antioxidantsagainst ionizing irradiation (Weiss et al. Radioprotection byAntioxidants. Ann N Y Acad Sci 2000; 899:44-60).

A new class of antioxidants, stable nitroxide radicals, has beensuggested as potent radioprotectors due to multiplicity of their directradical scavenging properties as well as catalytic enzyme-likemechanisms (Saito et al. Two reaction sites of a spin label, TEMPOL withhydroxyl radical. J Pharm Sci 2003; 92:275-280; Mitchell et al.Biologically active metal-independent superoxide dismutase mimics.Biochemistry 1990; 29:2802-2807). TEMPOL(4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a nitroxide whoseproperties as a radioprotector in vitro and in vivo have beenextensively studied (Mitchell et al. Nitroxides as radiation protectors.Mil Med 2002; 167:49-50; Hahn et al. In vivo radioprotection and effectson blood pressure of the stable free radical nitroxides. Int J RadiatOncol Biol Phys 1998; 42:839-842. Mitchell et al. Inhibition ofoxygen-dependent radiation-induced damage by the nitroxide superoxidedismutase mimic, tempol. Arch Biochem Biophys 1991; 289:62-70; Hahn etal. Tempol, a stable free radical, is a novel murine radiationprotector. Cancer Res 1992; 52:1750-1753). Currently, TEMPOL is inclinical trials as a topical radiation protector to prevent hair lossduring cancer radiotherapy. While found promising and relativelyeffective, the required high millimolar concentrations of TEMPOL, mainlydue to its poor partitioning into cells and mitochondria, set a limitfor its broader applications (Gariboldi et al. Study of in vitro and invivo effects of the piperidine nitroxide Tempol—a potential newtherapeutic agent for gliomas. Eur J Cancer 2003; 39:829-837). Inaddition, it has been demonstrated that TEMPOL must be present duringirradiation to exert its radioprotective effect (Mitchell et al.Radiation, radicals, and images. Ann N Y Acad Sci 2000; 899:28-43;Mitchell et al. Inhibition of oxygen-dependent radiation-induced damageby the nitroxide superoxide dismutase mimic, tempol. Arch BiochemBiophys 1991; 289:62-70), This suggests that the protective mechanismsof TEMPOL are limited to its interactions with short-lived radiolyticintermediates produced by irradiation.

Sufficient concentrations of antioxidants at the sites of free radicalgeneration are critical to optimized protection strategies. A great dealof research has indicated that mitochondria are both the primary sourceand major target of ROS (Reviewed in Orrenius S. Reactive oxygen speciesin mitochondria-mediated cell death. Drug Metab Rev 2007; 39:443-455).In fact, mitochondria have been long considered as an important targetfor drug discovery (Szewczyk et al., Mitochondria as a pharmacologicaltarget. 221 Pharmacol. Rev. 54:101-127; 2002; Garber K. Targetingmitochondria emerges as therapeutic strategy. J. Natl. Cancer Inst.97:1800-1801; 2005).

Chemistry-based approaches to targeting of compounds to mitochondriainclude the use of proteins and peptides, as well as the attachment ofpayloads to lipophilic cationic compounds, triphenyl phosphoniumphosphate, sulfonylureas, anthracyclines, and other agents with provenor hypothetical affinities for mitochondria (Murphy M P. Targetingbioactive compounds to mitochondria. Trends Biotechnol, 15:326-330;1997; Dhanasekaran et al., Mitochondria superoxide dismutase mimeticinhibits peroxideinduced oxidative damage and apoptosis: role ofmitochondrial superoxide. Free Radic. Biol. Med. 157 39:567-583; 2005;Hoye et al., Targeting Mitochondria. Ace. Chem. Res. 41: 87-97, 2008).However, at the time of this writing, no evidence has been presentedthat GS-nitroxides

SUMMARY

There remains a very real need for a composition and associated methodsfor delivering cargo of various types to mitochondria, specificallyantioxidants. Provided herein are compounds comprising a targeting groupand a cargo that is a nitroxide-containing group and compositionscomprising the compounds. As illustrated in the Examples, below,compounds and compositions described herein have use in the prophylaxisand treatment of exposure to ionizing radiation, in anti-ageingtherapies and, generally, in treating conditions that benefit fromantioxidant treatment. Examples of these compounds are provided belowand in the claims.

For example, the effective mitochondrial concentration of mitochondriatargeted conjugated nitroxides (—N-0., —N—OH or N═O containing compoundsand groups) against γ-irradiation could be increased up to 1,000 times(and their required tissues concentrations can be reduced 1,000 timesfrom 10 mM to 10 μM) compared with parent non-conjugated nitroxides.Enrichment in mitochondria of mitochondria targeted nitroxides has beendemonstrated by EPR spectroscopy as well as by MS analysis of theircontent in mitochondria obtained from cells incubated with mitochondriatargeted nitroxides. Delivery of mitochondria targeted-nitroxides intomitochondria does not depend on the mitochondrial membrane potential.Therefore, mitochondria targeted nitroxides can accumulate not only inintact but also in de-energized or damaged mitochondria with lowmembrane potential. Moreover, mitochondria targeted nitroxide conjugatesare delivered into mitochondria without affecting the mitochondrialmembrane potential. Hence, they do not impair the major mitochondrialfunction, the energy production, in cells. In addition, the conjugatednitroxides provide a new important feature, post irradiation protection.

Like other nitroxides, conjugated mitochondria targeted nitroxides mightpotentially lower blood pressure and sympathetic nerve activity.However, the dramatically reduced dose of mitochondria targetednitroxides (about 1,000-fold), compared to non-conjugated parentalnitroxides, may be significantly below of those inducing side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1A to 1-2G provide non-limiting examples of certain nitroxides.The log P values were estimated using the online calculator of molecularproperties and drug likeness on the Molinspirations Web site(www.molinspiration.com/cgi-bin/properties). TIPNO=tert-butyl isopropylphenyl nitroxide.

FIG. 2-1A to 2-3F provide examples of structures of certainmitochondria-targeting antioxidant compounds referenced herein, and thestructure of TEMPOL.

FIG. 3 depicts an example of a synthetic pathway for theTEMPO-hemigramicidin conjugates.

FIGS. 4A-4B show an EPR-based analysis of integration and reduction ofnitroxide Gramicidin S peptidyl-TEMPO conjugates in MECs.

FIGS. 5A-5H show a fluorescein isothiocyanate-dextran (FD4) read-outwhich reflects the effect of Gramicidin-S TEMPO conjugates on rat ilealmucosal permeability following profound hemorrhagic shock. Data areexpressed as a percentage of the change permeability relative to thatobserved in simultaneously assayed control segments loaded during shockwith normal saline solution. FIG. 5A shows an FD4 read-out of TEMPOLwhich is used as a “positive control” for the gut mucosal protectionassay. FIG. 5B shows an FD4 read-out of TEMPO conjugate XJB-5-208reflecting gut mucosal protection. FIG. 5C shows an FD4 read-out ofXJB-5-125 which has the TEMPO payload, but fails to provide protectionagainst gut barrier dysfunction induced by hemorrhage. FIG. 5D shows anFD4 read-out of XJB-5-127 which lacks the TEMPO payload and fails toprovide protection against gut barrier dysfunction induced byhemorrhage. FIG. 5E shows an FD4 read-out of TEMPO conjugate XJB-5-131reflecting gut mucosal protection. FIG. 5F shows an FD4 read-out ofXJB-5-133 which lacks the TEMPO payload even though it possesses thesame hemigramicidin mitochondria targeting moiety as the most activecompound, XJB-5-131. FIG. 5G shows an FD4 read-out of XJB-5-197 whichhas the TEMPO payload, but fails to provide protection against gutbarrier dysfunction induced by hemorrhage. FIG. 5H shows an FD4 read-outof XJB-5-194 which lacks the TEMPO payload and fails to provideprotection against gut barrier dysfunction induced by hemorrhage.

FIGS. 6A-6F show graphical representations of the effect of nitroxideconjugates on ActD-induced apoptosis. FIG. 6A is a graphicalrepresentation of superoxide production based upon mean fluorescenceintensity from 10,000 ileal cells. FIG. 6B is a graphical representationof phosphatidylserine (PS) externalization as indicated by thepercentage of annexin V-positive cells. FIG. 6C is a graphicalrepresentation of caspase-3 activity as indicated by amount of itsspecific substrate present, Z-DVED-AMC, in nmol/mg protein. FIG. 6D is agraphical representation of DNA fragmentation as indicated by propidiumiodide fluorescence. FIG. 6E is a graphical representation of PSexternalization at different concentrations of the compound 5a (as shownin FIG. 3). FIG. 6F is a graphical representation of adenosinetriphosphate (ATP) levels in mitochondria in the presence or absence of5a or 2-deoxyglucose.

FIG. 7 illustrates the effects of intraluminal XJB-5-131 onhemorrhage-induced peroxidation of phospholipids in intestinal mucosa.FIG. 7A is a graphical representation of the peroxidation ofphosphatidylcholine (“PC”). FIG. 7B is a graphical representation ofperoxidation activity with respect to phosphatidylethanolamine (“PE”).FIG. 7C is a graphical representation of peroxidation activity withrespect to phosphatidylserine (“PS”). FIG. 7D is a graphicalrepresentation of peroxidation activity with respect to cardiolipin(“CL”).

FIG. 8 is a graphical representation of caspase 3 and 7 activity thatillustrates the effects of intraluminal XJB-5-131.

FIG. 9 is a graphical representation of permeability of XJB-5-131 withrespect to Caco-2BBe human enterocyte-like monolayers subjected tooxidative stress. The permeability of the monolayers is expressed as aclearance (pL·h−1·cm2).

FIG. 10A is a graphical representation of the effects of intravenoustreatment with XJB-5-131 on MAP (mean arterial pressure, mm Hg) of ratessubjected to volume controlled hemorrhagic shock. FIG. 10B is agraphical representation of the effects of intravenous treatment withXJB-5-131 on survival probability of rates subjected to volumecontrolled hemorrhagic shock.

FIG. 11A is a schematic of a synthesis protocol for JP4-039. FIG. 11Bprovides a synthesis route for a compound of Formula 4, below.

FIGS. 12A-12B show that nitroxide conjugate XJB-5-125 integrates intocells and mitochondria much more efficiently than their parentnon-conjugated 4-amino-TEMPO in mouse embryonic cells. FIG. 12A showstheir cellular and mitochondrial integration efficiencies in mouseembryonic cells, and FIG. 12B shows representative EPR spectrum ofnitroxides recovered from mitochondria.

FIGS. 13A-13B reveal that nitroxide conjugate XJB-5-125 protects mouseembryonic cells against gamma irradiation induced superoxide generationand cardiolipin peroxidation. FIG. 13A superoxide generation. Cells wereexposed to 10 Gy of γ-irradiation. XJB-5-125 (20 μM) was added to cellseither 10-min before or 1-h after irradiation and removed after 5-hincubation. Cells were incubated with 5 μM DHE for 30 min at theindicated time points. Ethidium fluorescence was analyzed using aFACScan flow cytometer supplied with CellQuest software. Meanfluorescence intensity from 10,000 cells was acquired using a 585-nmbandpass filter. FIG. 13B Cardiolipin oxidation. Cardiolipinhydroperoxides were determined using a fluorescent HPLC-based Amplex Redassay. Data presented are means±S.E. (n=3). *p<0.01 vs non-irradiatedcells; *p<0.01(0.05) vs irradiated cells without XJB-5-125 treatmentunder the same condition. Insert is a typical 2D-HPTLC profile ofphospholipids from cells.

FIGS. 14A-14E reveal that nitroxide conjugate XJB-5-125 protects cellsagainst gamma irradiation induced apoptosis. FIG. 14A XJB-5-125 blocks7-irradiation induced accumulation of cytochrome c in the cytosol ofmouse embryonic cells. FIG. 14B Densitometry ratio of cytochromec/actin. Semi-quantitation of the bands was carried out by densitometryusing Labworks Image Acquisition and Analysis Software (UVP, Upland,Calif.). The level of cytochrome c release was expressed as the meandensitometry ratio of cytochrome c over actin. FIG. 14C Dose (5, 10 and20 μM) dependent radioprotective effect of XJB-5-125 (pre-treatment) onγ-irradiation (10 Gy) induced phosphatidylserine (PS) externalization.After 48 h post-irradiation incubation, cells were harvested and stainedwith annexin-V-FITC and propodium iodide (PI) prior to flow cytometryanalysis. FIG. 14D Time (2, 3, 4, 5, and 6 h) dependent radioprotectiveeffect of XJB-5-125 (201.04) on γ-irradiation (10 Gy) induced PSexternalization (48 h post irradiation) in mouse embryonic cells. FIG.14E Effect of XJB-5-125 on γ-irradiation (10 Gy) induced PSexternalization in human bronchial epithelial cell line BEAS-2B cells.Cells were treated with 5-125 (5 or 10 μM) before (10-min) or after(1-h) irradiation. Externalization of PS was analyzed 72 hpost-irradiation exposure. Data shown are means±S.E. (n=3).*(&)p<0.01(0.05) vs irradiated cells without 5-125 treatment, # p<0.05vs cells pre-treated with 5-125.

FIG. 15 shows the effect of nitroxide conjugate XJB-5-125 ongamma-irradiation dose survival curves of mouse embryonic cells. Cellswere pre- (10-min) or post-treated (1-h) with XJB-5-125 (20 μM), whichwas removed after 4-h incubation period. The surviving fraction wascalculated as the plating efficiency of the samples relative to that ofthe control. The data was fitted to a single-hit multitarget model usingSigmaPlot 9.0 (Systat Software). Data presented are the mean±S.E. (n=3).

FIG. 16 illustrates the effect of GS conjugated nitroxide, XJB-5-125, ongamma-irradiation dose survival curves of 32D cl 3 murine hematopoieticcells. The cells incubated in XJB-5-125 or Tempol had an increased Do(1.138 or 1.209 Gy, respectively) compared to the 32D cl 3 cells (0.797Gy). The cells incubated in XJB-5-125 had an increased shoulder on thesurvival curve with an n of 18.24 compared to 5.82 for the cellsincubated in tempol.

FIGS. 17A and 17B are graphs showing GS-nitroxide compound JP4-039increases survival of mice exposed to 9.75 Gy total body irradiation.

FIG. 18 is a graph showing that GS-nitroxide compound JP4-039 increasessurvival of mice exposed to 9.5 Gy total body irradiation.

FIG. 19 is a graph showing that GS-nitroxide JP4-039 is an effectivehematopoietic cell radiation mitigator when delivered 24 hr afterirradiation.

FIG. 20 is a graph showing that JP4-039 is an effective mitigator ofirradiation damage to KM101 human marrow stromal cells.

FIGS. 21A-21B show results with detection of human cells in NOD/SCIDmouse marrow harvested 27 days after cord blood transplanted I.V,showing flow cytometric analysis and identification of human CD45+(lightgray) hematopoietic cells in NOD/SCED mouse BM following irradiation,proximal tibia bone drilling (see below), and human cord bloodinjection.

FIG. 21C is a photomicrograph of cross-section through a tibial wound7-days after surgical construction with a drill bit of a unicortical2-mm diameter wound in the lateral aspect of the tibia 2-mm below theproximal epiphyseal plate.

FIG. 22 is a schematic diagram of a Bronaugh diffusion system forstudying in vitro transdermal flux.

FIG. 23 is a graph showing delivery of XJB-5-125 into mouse skin after24 hours.

FIGS. 24A-24D show typical EPR spectra of GS-nitroxides recorded fromdifferent fractions obtained after the filtration through the mouseskin. 1—donor fluid, 2—receiver fluid after 6 h of solution Afiltration, 3—receiver fluid after 6 h of solution B filtration, 4—skinafter 24 h exposure to solution A. The EPR spectra of GS-nitroxideradicals in medium, or skin homogenates were recorded in 28.5% ofacetonitrile with addition of 2 mM K₃Fe(CN)₆

FIG. 25 is a graph showing cumulative transdermal absorption ofXJB-5-125 through mouse skin over 24 hours

FIGS. 26A and 26B provide structures for compounds JED-E71-37 andJED-E71-58, respectively.

FIG. 27 shows a treatment paradigm for study to determine the impact ofXJB-5-131 on the age at onset of signs of aging in progeroid Ercc1^(−/Δ)mice. XJB-5-131 was 2 mg/kg prepared from a 10 μg/μL stock in DMSO mixedwith 50 μL, of sunflower seed oil and injected intraperitoneally. Ascontrol littermate Ercc1^(−/Δ) mice were treated with an equal volume ofsunflower seed oil only, in double-blind twin study.

FIG. 28 is a summary table showing effects of treatment with XJB-5-131(“XJB” in this figure), relative to control (sunflower seed oil) on theage at onset (in weeks) of various indicia of aging in Ercc1^(−/Δ) mice,using the protocol of FIG. 27. The duration of treatment of mice in thisfigure was three times per week, beginning at 5 wks of age andcontinuing throughout their lifespan. Cells highlighted in the XJBcolumn indicate a significant delay in onset of the age-relateddegenerative change in mice treated with XJB relative to isogeniccontrols treated with vehicle only.

FIG. 29 is a bar graph showing glycosaminoglycan (an extracellularmatrix protein that is essential for disc maintenance and flexibility)content of intervertebral discs of Ercc1^(−/Δ) mice either treated withXJB-5-131 (“XJB” in this figure) or vehicle (sunflower seed oil)according to the protocol shown in FIG. 27. The duration of treatment ofmice in this figure was three times per week, beginning at 5 wks of ageand continuing throughout their lifespan.

FIG. 30 provides photographs showing the effects of (photo)aging inErcc1^(−/Δ) mice either treated with XJB-5-131 (80 μg emulsified in atopical cream) or cream only, according to the protocol shown in FIG.27. The duration of treatment of mice in this figure was daily for fivedays post-UV irradiation.

FIGS. 31A-B are graphs showing weights as a function of age of FIG. 31Amale and FIG. 31B female Ercc1^(−/Δ) mice either treated with XJB-5-131or vehicle (sunflower seed oil) according to the protocol shown in FIG.27. XJB-5-131 does not cause weight loss as does the parental compoundTEMPO.

FIG. 32 provides photomicrographs of SA-β galactosidase (a marker ofcellular senescence) staining in mouse embryonic fibroblast (“MEF”)cells prepared from Ercc1^(−/−) mice, where the MEF cells were eithertreated with XJB-5-131 (“XJB” in this figure; 500 nM dissolved in media)or media alone continuously for 48 hr prior to fixing and staining thecells.

FIG. 33 provides photomicrographs of γH2ΛX immunostaining (a marker ofDNA double strand breaks and cellular senescence) of mouse embryonicfibroblast (“MEF”) cells prepared from Ercc1^(−/−) mice, where the MEFcells were either treated with XJB-5-131 (“XJB” in this figure; 500 nMdissolved in media) or media alone continuously for 48 hr prior tofixing and staining the cells.

FIG. 34 is a graph showing apoptosis in mouse embryonic fibroblast(“MEF”) cells prepared from Ercc1^(−/−) mice, where the MEF cells wereeither treated with XJB-5-131 (“XJB” in this figure; 500 nM dissolved inmedia) or media alone continuously for 48 hr prior to fixing andstaining the cells.

FIG. 35 provides photomicrographs showing the effects of varying dosesof JP4-039 on proliferation and growth of mouse embryonic fibroblast(“MEP”) cells prepared from Ercc1^(−/−) mice. JP4-039 is not toxic tocells at concentrations as high as 10 μM.

FIG. 36 provides photomicrographs showing the effects of varying dosesof JP4-039 on proliferation and growth of mouse embryonic fibroblast(“MEF”) cells prepared from wild-type mice. JP4-039 is not toxic tocells at concentrations as high as 10 μM.

FIG. 37 provides photomicrographs showing levels of p16, a marker ofirreversible cellular senescence, in mouse embryonic fibroblast (“MEF”)cells prepared from Ercc1^(−/Δ) mice, where the MEF cells were eithertreated with either JP4-039 (“0-39” in this figure; 10 μM dissolved inmedia) or media alone for 48 hrs prior to fixing and immunostaining thecells.

FIG. 38 provides photomicrographs showing cell proliferation of primarymouse embryonic fibroblast (“MEF”) cells prepared from Ercc1^(−/−) miceand grown in conditions of oxidative stress (20% oxygen), where the MEFcells were either treated with either JED-E71-37, JED-E71-58 91 uMdissolved in media), or media alone for a duration of 48 hrs prior tofixing and staining the cells.

FIG. 39 provides photomicrographs showing γH2AX immunostaining (a markerof DNA double strand breaks and cellular senescence) of mouse embryonicfibroblast (“MEF”) cells prepared from Ercc1^(−−Δ) mice and grown inconditions of oxidative stress (20% oxygen), where the MEF cells wereeither treated with either JED-E71-58 (1 μM dissolved in media, or mediaalone for a duration of 48 hrs prior to fixing and staining the cells.

FIG. 40 is a schematic showing alternative designs of nitroxideanalogues.

FIG. 41 is a schematic of a synthesis protocol for various alternativedesigns of nitroxide analogues.

FIG. 42 is a schematic of a synthesis protocol for an alternativenitroxide moiety of 1,133-tetramethylisoindolin-2-yloxyl (TMIO).

FIG. 43 is a schematic of a synthesis protocol for an alternativenitroxide moiety of 1-methyl 2-azaadamantane N-oxyl (1-Me-AZADO).

DETAILED DESCRIPTION

As used herein, the term “subject” refers to members of the animalkingdom including but not limited to human beings. The term “reactiveoxygen species” (“ROS”) includes, but is not limited to, superoxideanion, hydroxyl, and hydroperoxide radicals.

An antioxidant compound is defined herein as a compound that decreasesthe rate of oxidation of other compounds or prevents a substance fromreacting with oxygen or oxygen containing compounds. A compound may bedetermined to be an antioxidant compound by assessing its ability todecrease molecular oxidation and/or cellular sequellae of oxidativestress, for example, and without limitation, the ability to decreaselipid peroxidation and/or decrease oxidative damage to protein ornucleic acid. In one embodiment, an antioxidant has a level ofantioxidant activity between 0.01 and 1000 times the antioxidantactivity of ascorbic acid in at least one assay that measuresantioxidant activity.

Provided herein are compounds and compositions comprising a targetinggroup and a cargo, such as a nitroxide-containing group. The cargo maybe any useful compound, such as an antioxidant, as are well known in themedical and chemical arts. The cargo may comprise a factor havinganti-microbial activity. For example, the targeting groups may becross-linked to antibacterial enzymes, such as lysozyme, or antibiotics,such as penicillin. Other methods for attaching the targeting groups toa cargo are well known in the art. In one embodiment, the cargo is anantioxidant, such as a nitroxide-containing group. In anotherembodiment, the cargo transported by mitochondria-selective targetingagents may include an inhibitor of NOS activity. The cargo may have aproperty selected from the group consisting of antioxidant,radioprotective, protective, anti-apoptotic, therapeutic, ameliorative,NOS antagonist and combinations thereof. In another embodiment, thecargo may have the ability to inhibit nitric oxide synthase enzymeactivity. It will be appreciated that a wide variety of cargos may beemployed in the composition described herein. Non-limiting examples ofcargos include: a 2-amino-6-methyl-thiazine, a ubiquinone analog, aubiquinone analog fragment moiety, a ubiquinone analog fragment moietylacking a hydrophilic tail, a superoxide dismutase mimetic, a superoxidedismutase biomimetic and a salen-manganese compound.

While the generation of ROS in small amounts is a typical byproduct ofthe cellular respiration pathway, certain conditions, including adisease or other medical condition, may occur in the patient when theamount of ROS is excessive to the point where natural enzyme mechanismscannot scavenge the amount of ROS being produced. Therefore, compounds,compositions and methods that scavenge reactive oxygen species that arepresent within the mitochondrial membrane of the cell are useful and areprovided herein. These compounds, compositions and methods have theutility of being able to scavenge an excess amount of ROS being producedthat naturally occurring enzymes SOD and catalase, among others, cannotcope with.

In one non-limiting embodiment, the compound has the structure:

wherein X is one of

andR₁, R₂ and R₄ are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including:methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl. R₃ is—NH—R₅, —O—R₅ or —CH₂—R₅, where R₅ is an —N—O., —N—OH or N═O containinggroup. In one embodiment, R₃ is

(I-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another embodimentR₃is

(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl).R is —C(O)—R₆, —C(O)O—R₆, or —P(O)—(R₆)₂, wherein R₆ is C₁-C₆ straightor branched-chain alkyl optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Ac (Acetyl, R═—C(O)—CH₃), Boc(R═—C(O)O-tert-butyl), Cbz (R═—C(O)O-benzyl (Bn)) groups. R also may bea diphenylphosphate group, that is,

Excluded from this is the enantiomer XJB-5-208. In certain embodiments,R₁ is t-butyl and R₂ and R₄ are H; for instance:

As used herein, unless indicated otherwise, for instance in a structure,all compounds and/or structures described herein comprise all possiblestereoisomers, individually or mixtures thereof.

As indicated above, R₅ is an —N—O., —N—OH or —N═O containing group (not—N—O., —N—OH or —N═O alone, but groups containing those moieties, suchas TEMPO, etc, as described herein). As is known to one ordinarilyskilled in the art, nitroxide and nitroxide derivatives, includingTEMPOL and associated TEMPO derivatives are stable radicals that canwithstand biological environments. Therefore, the presence of the4-amino-TEMPO, TEMPOL or another nitroxide “payload” within themitochondria membrane can serve as an effective and efficient electronscavenger of the ROS being produced within the membrane. Non-limitingexamples of this include TEMPO (2,2,6,6-Tetramethyl-4-piperidine 1-oxyl)and TEMPOL (4-Hydroxy-TEMPO), in which, when incorporated into thecompound described herein, for example, when R₃ is —NH—R₅, —O—R₅:

Additional non-limiting examples of —N—O®, —N—OH or N═O containing groupare provided in Table 1 and in FIG. 1 (from Jiang, J., et al.“Structural Requirements for Optimized Delivery, Inhibition of OxidativeStress, and Antiapoptotic Activity of Targeted Nitroxides”, J PharmacolExp Therap. 2007, 320(3):1050-60). A person of ordinary skill in the artwould be able to conjugate (covalently attach) any of these compounds tothe rest of the compound using common linkers and/or conjugationchemistries, such as the chemistries described herein. Table 1 providesa non-limiting excerpt from a list of over 300 identifiedcommercially-available —N—O., —N—OH or N═O containing compounds that maybe useful in preparation of the compounds or compositions describedherein.

TABLE 1 Commercially-available —N—O•, —N—OH or N═O containing groupsStructure Name CAS No.

Trimethylamine N-Oxide 1184-78-7

N,N-Dimethyldodecylamine N-Oxide 1643-20- 570592-80-2

N-Benzoyl-N- Phenylhydroxylamine 304-88-1

N,N-Diethylhydroxylamine 3710-84-7

N,N- Dibenylhydroxylamine 14165-27- 6621-07-8

Di-Tert-Butyl Nitroxide 2406-25-9

N,N- Dimethylhydroxylamine Hydrochloride 16645-06-0

Metobromuron 3060-89-7

Benzyl-Di-Beta-Hydroxy Ethylamine-N-Oxide

Bis(Trifluoromethyl)Nitroxide 2154-71-4

Triethylamine N-Oxide 2687-45-8

N-Methoxy-N- Methylcarbamate 6919-62-6

N,N-Bis(2-Chloro-6- Fluorobenzyl)-N-[(([2,2- Dichloro-1-(1,4-Thiazinan-4-yl+)ethylidene] amino)carbonyl)oxy]amine

Tri-N-Octylamine N-Oxide 13103-04-3

Diethyl (N-Methoxy-N- Methylcarbamoylmethyl)Phos- phonate 124931-12- 0

N-Methoxy-N-Methyl-2- (Triphenylphosphoranylidene) Acetamide 129986-67-0

N-Methoxy-N-Methyl-N′- [5-Oxo-2- (Trifluoromethyl)-5h-Chromeno[2,3-B]Pyridi+ N-3-yl]Urea

N-[(4-Chlorobenzyl)Oxy]- N-([5-Oxo-2-Phenyl-1,3- Oxazol-4(5h)-Yliden]Methyl+N Acetamide

N-Methylfurohydroxamic Acid 109531-96- 6

N,N-Dimethylnonylamine N-Oxide 2536-13-2

N-(Tert-Butoxycarbonyl)-L- Alanine N′-Methoxy-N′- Methylamide 87694-49-3

1-(4-Bromophenyl)-3- (Methyl([3- (Trifluoromethyl)Benzoyl]Oxy)Amino)-2-Prop+ En-1- One

2- ([[(Anilinocarbonyl)Oxy](Meth- yl)Amino]Methylene)-5-(4-Chlorophenyl)-1,3+- Cyclohexanedione

N-Methoxy-N-Methyl-2- (Trifluoromethyl)-1,8- Naphthyridine-3-Carboxamide

N-Methoxy-N-Methyl- Indole-6-Carboxamide

AKOS 91254 127408-31- 5

N-[(3s,4r)-6-Cyano-3,4- Dihydro-3-Hydroxy-2,2- Dimethyl-2h-1-Benzopyran-4-Y+ L]-N-Hydroxyacetamide 127408-31- 5

N-Methoxy-N-Methyl-1,2- Dihydro-4-Oxo- Pyrrolo[3,2,1-1j]Quinoline-5-Carboxa+ Mide

Fr-900098

2,2′-(Hydroxyimino)Bis- Ethanesulfonic Acid Disodium Salt 133986-51- 3

Fmoc-N-Ethyl- Hydroxylamine

Bis(N,N- Dimethylhydroxamido)Hydrox- ooxovanadate

Pyraclostrobin 175013-18- 0

1-Boc-5-Chloro-3- (Methoxy-Methyl- Carbamoyl)Indazole

N-Methoxy-N-Methyl- Thiazole-2-Carboxamide

4,4-Difluoro-N-Methyl-N- Methoxy-L-Prolinamide Hcl

3-Fluoro-4- (Methoxy(Methyl)Carbamoyl) Phenylboronic Acid 913835-59- 3

1-Isopropyl-N-Methoxy-N- Methyl-1h- Benzo[D][1,2,3]Triazole-6-Carboxamide 467235-06- 9

(Trans)-2-(4-Chlorophenyl)- N-Methoxy-N- Methylcyclopropanecarboxa- mide

Bicyclo[2.2.1]Heptane-2- Carboxylic Acid Methoxy- Methyl-Amide

Akos Bc-0582

3-(N,O- Dimethylhydroxylaminocarbo- nyl)Phenylboronic Acid, PinacolEster

1-Triisopropylsilanyl-1h- Pyrrolo[2,3-B]Pyridine-5- Carboxylic AcidMethoxy+- Methyl-Amide

According to one embodiment, the compound has the structure

or the structure

wherein R is —NH—R₁, —O—R₁ or —CH₂—R₁, and R₁ is an —N—O., —N—OH or N═O)containing group. In one embodiment, R is —NH—R₁, and in another R is—NH-TEMPO.

According to another embodiment, the compound has the structure:

in which R1, R2 and R3 are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including2-methyl propyl, benzyl, methyl-, hydroxyl- or fluoro-substitutedbenzyl, such as 4-hydroxybenzyl. R4 is an —N—O., —N—OH or N═O containinggroup. In one embodiment, R4 is

(1-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another embodimentR4 is

(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is —C(O)—R5,—C(O)O—R5, or —P(O)—(R5)₂, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Ac, Boc, and Cbz groups. R also may be adiphenylphosphate group, that is

In certain specific embodiments, in which R4 is TEMPO, the compound hasone of the structures A, A1, A2, or A3 (Ac=Acetyl=CH₃C(O)—):

-   -   According to another embodiment, the compound has the structure

In which R1, R2 and R3 are, independently, hydrogen, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including2-methyl propyl, benzyl, methyl-, hydroxyl- or fluoro-substitutedbenzyl, such as 4-hydroxybenzyl. R4 is an —N—O., —N—OH or N═O containinggroup. In one embodiment, R4 is

(1-Me-AZADO or 1-methyl 2-azaadamantane N-oxyl). In another embodimentR4 is

(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl). R is —C(O)—R5,—C(O)O—R5, or —P(O)—(R5)₂, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Ac, Boc, and Cbz groups. R also may be adiphenylphosphate group, that is, R=

In certain specific embodiments, in which R4 is TEMPO, the compound hasone of the structures D, D1, D2, or D3 (Ac=Acetyl=CH₃C(O)—):

In another non-limiting embodiment, the compound has the structure:

wherein X is one of

L and

R₁ and R₄ are, independently, hydrogen, C₁-C₆ straight or branched-chainalkyl, optionally including a phenyl (C₆H₅) group, that optionally ismethyl-, hydroxyl- or fluoro-substituted, including: methyl, ethyl,propyl, 2-propyl, butyl, l-butyl, pentyl, hexyl, benzyl, hydroxybenzyl(e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl. R₃ is —NH—R₅, —O—R₅or —CH₂—R₅, where R₅ is an —N—O., —N—OH or N═O containing group. In oneembodiment, R₃ is

(1-Me-AZADO or 1-methyl azaadamantane N-oxyl). In another embodiment R₃is

(TMIO; 1,1,3,3-tetramethylisoindolin-2-yloxyl).R is —C(O)—R₆, —C(O)O—R₆, or —P(O)—(R₆)₂, wherein R₆ is C₁-C₆ straightor branched-chain alkyl optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Ac (Acetyl, R═—C(O)—CH₃), Boc(R═—C(O)O-tert-butyl), Cbz (R═—C(O)O-benzyl (Bn)) groups. R also may bea diphenylphosphate group, that is.

In one non-limiting embodiment, the compound has one of the structures

In yet another non-limiting embodiment, the compound has the structure

in which R₄ is hydrogen or methyl.

The compounds described above, such as the compound of Formula 1, can besynthesized by any useful method. The compound JP4-039 was synthesizedby the method of Example 8. In one embodiment, a method of making acompound of Formula 1 is provided. The compounds are synthesized by thefollowing steps:

reacting an aldehyde of structure R₁—C(O)—, wherein, for example andwithout limitation, R₁ is C₁-C₆ straight or branched-chain alkyl,optionally including a phenyl (C₆H₅) group, that optionally is methyl-,hydroxyl- or fluoro-substituted, including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, with(R)-2-methylpropane-2-sulfinamide to form an inline, for example

reacting a terminal alkyne-1-ol (HCC—R₂—CH₂—OH), wherein, for exampleand without limitation, R₂ is not present or is branched orstraight-chained alkylene, including methyl, ethyl, propyl, etc., with atert-butyl diphenylsilane salt to produce an alkyne, for example

reacting (by hydrozirconation) the alkyne with the imine in the presenceof an organozirconium catalyst to produce an alkene, for example

D1. acylating the alkene to produce a carbamate, for example

wherein, for example and without limitation, R₁ is C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, includingincluding: methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl,hexyl, benzyl, hydroxybenzyl (e.g., 4-hydroxy benzyl), phenyl andhydroxyphenyl;

D2. optionally, cyclopropanating the alkene and then acylating thealkene to produce a carbamate, for example

wherein, for example and without limitation, R₃ is C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, includingincluding: methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl,hexyl, benzyl, hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl andhydroxyphenyl;removing the t-butyldiphenylsilyl group from the carbamate to produce analcohol, for example

oxidizing the alcohol to produce a carboxylic acid, for example

andreacting the carboxylic acid with a nitroxide-containing compoundcomprising one of a hydroxyl or amine in a condensation reaction toproduce the antioxidant compound, for example

wherein R₄ is —NH—R₄ or —O—R₄, and R, is an —N—O., —N—OH or N═Ocontaining group, such as described above.

In another non-limiting embodiment, a compound is provided having thestructure R1-R2-R3 in which R1 and R3 are a group having the structure—R4-R5, in which R4 is a mitochondria targeting group and R5 is —NH—R6,—O—R6 or —CH₂—R6, wherein R6 is an —N—O., —N—OH or N═O containing group,such as TEMPO. R1 and R2 may be the same or different. Likewise, R4 andR5 for each of R1 and R3 may be the same or different. R2 is a linkerthat, in one non-limiting embodiment, is symmetrical. FIGS. 26A and 26Bdepicts two examples of such compounds. In one embodiment, R1 and R2have the structure shown in formulas 1, 2, or 3, above, with all groupsas defined above, including structures A, A1, A2, A3, D, D1, D2 and D3,above, an example of which is compound JED-E71-58, shown in FIG. 26B. Inanother embodiment, R1 and R2 are, independently, a gramicidinderivative, for example, as in the compound JED-E71-37, shown in FIG.26A. Examples of gramicidin derivatives are provided herein, such asXJB-5-131 and XJB-5-125 (see. FIG. 2), and are further described bothstructurally and functionally in United States Patent Publication Nos.20070161573 and 20070161544 as well as in Jiang, J, et al. (StructuralRequirements for Optimized Delivery, Inhibition of Oxidative Stress, andAntiapoptotic Activity of Targeted Nitroxides, J Pharmacol Exp Therap.2007, 320(3):1050-60, see also, Hoye, A T et al., TargetingMitochondria, Acc Chem Res. 2008, 41(1):87-97, see also, Wipf, P, etal., Mitochondrial Targeting of Selective Electron Scavengers: Synthesisand Biological Analysis of Hemigramicidin-TEMPO Conjugates, (2005) J AmChem Soc. 2005, 127:12460-12461). The XJB compounds can be linked into adimer, for example and without limitation, by reaction with the nitrogenof the BocHN groups (e.g., as in XJB-5-131), or with an amine, ifpresent, for instance, if one or more amine groups of the compound isnot acylated to form an amide (such as NHBoc or NHCbx).

In Jiang, J, et al. (J Pharmacol Exp Therap. 2007, 320(3):1050-60),using a model of ActD-induced apoptosis in mouse embryonic cells, theauthors screened a library of nitroxides to explore structure-activityrelationships between their antioxidant/antiapoptotic properties andchemical composition and three-dimensional (3D) structure. Highhydrophobicity and effective mitochondrial integration were deemednecessary but not sufficient for high antiapoptotic/antioxidant activityof a nitroxide conjugate. By designing conformationally preorganizedpeptidyl nitroxide conjugates and characterizing their 3D structureexperimentally (circular dichroism and NMR) and theoretically (moleculardynamics), they established that the presence of the β-turn/β-sheetsecondary structure is essential for the desired activity. Monte Carlosimulations in model lipid membranes confirmed that the conservation ofthe D-Phe-Pto reverse turn in hemi-GS analogs ensures the specificpositioning of the nitroxide moiety at the mitochondrial membraneinterface and maximizes their protective effects. These insights intothe structure-activity relationships of nitroxide-peptide and -peptideisostere conjugates are helpful in the development of newmechanism-based therapeutically effective agents, such as thosedescribed herein.

Targeting group R4 may be a membrane active peptide fragment derivedfrom an antibiotic molecule that acts by targeting the bacterial cellwall. Examples of such antibiotics include: bacitracins, gramicidins,valinomycins, enniatins, alamethicins, beauvericin, serratomolide,sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinumpeptides. The membrane-active peptide fragment derived from anantibiotic may include the complete antibiotic polypeptide, or portionsthereof having membrane, and preferably mitochondria-targetingabilities, which is readily determined, for example, by cellularpartitioning experiments using radiolabeled peptides Examples of usefulgramicidin-derived membrane active peptide fragments are theLeu-D-Phe-Pro-Val-Orn and D-Phe-Pro-Val-Orn-Leu hemigramicidinfragments. As gramicidin is cyclic, any hemigramicidin 5-mer is expectedto be useful as a membrane active peptide fragment, includingLeu-D-Phe-Pro-Val-Orn, D-Phe-Pro-Val-Orn-Leu, Pro-Val-Orn-Leu-D-Phe,Val-Orn-Leu-D-Phe-Pro and Orn-Leu-D-Phe-Pro-Val (from Gramicidin S). Anylarger or smaller fragment of gramicidin, or even larger fragmentscontaining repeated gramicidin sequences (e.g.,Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro) are expectedto be useful for membrane targeting, and can readily tested for suchactivity, in one embodiment, the Gramicidin S-derived peptide comprisesa β-turn, which appears to confer to the peptide a high affinity formitochondria. Derivatives of Gramicidin, or other antibiotic fragments,include isosteres (molecules or ions with the same number of atoms andthe same number of valence electrons—as a result, they can exhibitsimilar pharmacokinetic and pharmacodynamic properties), such as(E)-alkene isosteres (see. United Stales Patent Publication Nos.20070161573 and 20070161544 for exemplary synthesis methods). As withGramicidin, the structure (amino acid sequence) of bacitracins, oiliergramicidins, valinomycins, enniatins, alamethicins, beauvericin,serratomolide, sporidesmolide, tyrocidins, polymyxins, monamycins, andlissoclinum peptides are all known, and fragments of these can bereadily prepared and their membrane-targeting abilities can easily beconfirmed by a person of ordinary skill in the art.

Alkene isosteres such as (E)-alkene isosteres of Gramicidin S (i.e.,hemigramicidin) were used as part of the targeting sequence. See FIG. 3for a synthetic pathway for (E)-alkene isosteres and reference number 2for the corresponding chemical structure. First, hydrozirconation ofalkyne (FIG. 3, compound 1) with Cp₂ZrHCl is followed by transmetalationto Me₂Zn and the addition of N-Boc-isovaleraldimine. The resultingcompound (not shown) was then worked up using a solution oftetrabutylammonium fluoride (“TBAF”) and diethyl ether with a 74% yield.The resulting compound was then treated with acetic anhydride,triethylamine (TEA), and 4-N,N1-(dimethylamino) pyridine (“DMAP”) toprovide a mixture of diastereomeric allylic amides with a 94% yieldwhich was separated by chromatography. Finally, the product was workedup with K₂CO₃ in methanol to yield the (E)-alkene, depicted as compound2. The (E)-alkene, depicted as compound 2 of FIG. 3, was then oxidizedin a multi-step process to yield the compound 3 (FIG. 3)—an example ofthe (E)-alkene isostere.

The compound 3 of FIG. 3 was then conjugated with the peptideH-Pro-Val-Orn (Cbz)-OMe using 1-ethyl-3-(3-dimethylaminopropylcarbodiimide hydrochloride) (EDC) as a coupling agent. The peptide is anexample of a suitable targeting sequence having affinity for themitochondria of a cell. The resulting product is shown as compound 4a inFIG. 3. Saponification of compound 4a followed by coupling with4-amino-TEMPO (4-AT) afforded the resulting conjugate shown as compound5a in FIG. 3, in which the Leu-DPhe peptide bond has been replaced withan (E)-alkene.

In an alternate embodiment, conjugates 5 b in FIG. 3 was prepared bysaponification and coupling of the peptide 4b(Boc-Leu-DPhe-Pro-Val-Orn(Cbz)-OMe) with 4-AT. Similarly, conjugate 5cin FIG. 3 was prepared by coupling the (E)-alkene isostere as indicatedas compound 3 in FIG. 3 with 4-AT. These peptide and peptide analogs areadditional examples of suitable targeting sequences having an affinityto the mitochondria of a cell.

In another embodiment, peptide isosteres may be employed as theconjugate. Among the suitable peptide isosteres are trisubstituted(E)-alkene peptide isosteres and cyclopropane peptide isosteres, as wellas all imine addition products of hydro- or carbometalated internal andterminal alkynes for the synthesis of d-i and trisubstituted (E)-alkeneand cyclopropane peptide isosteres. See Wipf et al. Imine additions ofinternal alkynes for the synthesis of trisubstituted (E)-alkene andcyclopropane isosteres. Adv Synth Catal. 2005, 347:1605-1613. Thesepeptide mimetics have been found to act as β-turn promoters. See Wipf etal. Convergent Approach to (E)-Alkene and Cyclopropane PeptideIsosteres, Org Lett. 2005, 7(1):103-106.

The linker, R2, may be any useful linker, chosen for its active groups,e.g., carboxyl, alkoxyl, amino, sulfhydryl, amide, etc. Typically, asidefrom the active groups, the remainder is non-reactive (such as saturatedalkyl or phenyl), and does not interfere, sterically or by any otherphysical or chemical attribute, such as polarity orhydrophobicity/hydrophilicity, in a negative (loss of function) capacitywith the activity of the overall compound. In one embodiment, aside fromthe active groups, the linker comprises a linear or branched saturatedC₄-C₂₀ alkyl. In one embodiment, the linker. R2 has the structure

In which n is 4-18, including all integers therebetween, in oneembodiment, 8-12, and in another embodiment, 10.

A person skilled in the organic synthesis arts can synthesize thesecompounds by crosslinking groups R1 and R3 by any of the manychemistries available. In one embodiment, R1 and R3 are to R2 by anamide linkage (peptide bond) formed by dehydration synthesis(condensation) of terminal carboxyl groups on the linker and an amine onR1 and R3 (or vice versa). In one embodiment, R1 and R3 are identical ordifferent and are selected from the group consisting of: XJB-5-131,XJB-5-125, XJB-7-75, XJB-2-70, XJB-2-300, XJB-5-208, XJB-5-197,XJB-5-194, JP4-039 and JP4-049, attached in the manner shown in FIGS.26A and 26B.

In a therapeutic embodiment, a method of scavenging free-radicals in asubject (e.g., a patient in need of treatment with a free-radicalscavenger) is provided, comprising administering to the subject anamount of one or more compound described herein and having afree-radical scavenging group, such as a nitroxide-containing groupeffective to scavenge free radicals. As described above, a number ofdiseases, conditions or in juries can be ameliorated or otherwisetreated or prevented by administration of free radical scavengingcompounds, such as those described herein.

In any case, as used herein, any agent or agents used for prevention,mitigation or treatment in a subject of injury caused by radiationexposure is administered in an amount effective to prevent, mitigate oftreat such injury, namely in an amount and in a dosage regimen effectiveto prevent injury or to reduce the duration and/or severity of theinjury resulting from radiation exposure. According to one non-limitingembodiment, an effective dose ranges from 0.1 or 1 mg/kg to 100 mg/kg,including any increment or range therebetween, including 1 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 50 mg/kg, and 75 mg/kg. However,for each compound described herein, an effective dose or dose range isexpected to vary from that of other compounds described herein for anynumber of reasons, including the molecular weight of the compound,bioavailability, specific activity, etc. For example and withoutlimitation, where XJB-5-131 is the antioxidant, the dose may be betweenabout 0.1 and 20 mg/kg, or between about 0.3 and 10 mg/kg, or betweenabout 2 and 8 mg/kg, or about 2 mg/kg and where either JP4-039,JED-E71-37 or JED-E71-58 is the antioxidant, the dose may be betweenabout 0.01 and 50 mg/kg, or between about 0.1 and 20 mg/kg, or betweenabout 0.3 and 10 mg/kg, or between about 2 and 8 mg/kg, or about 2mg/kg. The therapeutic window between the minimally-effective dose, andmaximum tolerable dose in a subject can be determined empirically by aperson of skill in the art, with end points being determinable by invitro and in vivo assays, such as those described herein and/or areacceptable in the pharmaceutical and medical arts for obtaining suchinformation regarding radioprotective agents. Different concentrationsof the agents described herein are expected to achieve similar results,with the drug product administered, for example and without limitation,once prior to an expected radiation dose, such as prior to radiationtherapy or diagnostic exposure to ionizing radiation, during exposure toradiation, or after exposure in any effective dosage regimen. Thecompounds can be administered continuously, such as intravenously, oneor more times daily, once every two, three, four, five or more days,weekly, monthly, etc., including increments therebetween. A person ofordinary skill in the pharmaceutical and medical arts will appreciatethat it will be a matter of simple design choice and optimization toidentify a suitable dosage regimen for prevention, mitigation ortreatment of injury due to exposure to radiation.

The compounds described herein also are useful in preventing, mitigating(to make less severe) and/or treating injury caused by radiationexposure. By radiation, in the context of this disclosure, it is meanttypes of radiation that result in the generation of free radicals, e.g.,reactive oxygen species (ROS), as described herein. The free radicalsare produced, for example and without limitation, by direct action ofthe radiation, as a physiological response to the radiation and/or as aconsequence of damage/injury caused by the radiation. In one embodimentthe radiation is ionizing radiation. Ionizing radiation consists ofhighly-energetic particles or waves that can detach (ionize) at leastone electron from an atom or molecule. Examples of ionizing radiationare energetic beta particles, neutrons, and alpha particles. The abilityof light waves (photons) to ionize an atom or molecule varies across theelectromagnetic spectrum. X-rays and gamma rays can ionize almost anymolecule or atom; far ultraviolet light can ionize many atoms andmolecules; near ultraviolet and visible light are ionizing to very fewmolecules. Microwaves and radio waves typically are considered to benon-ionizing radiation, though damage caused by, e.g., microwaves, mayresult in the production of free-radicals as part of the injury and/orphysiological response to the injury.

The compounds typically are administered in an amount and dosage regimento prevent, mitigate or treat the effects of exposure of a subject toradiation. The compounds may be administered in any manner that iseffective to treat, mitigate or prevent damage caused by the radiation.Examples of delivery routes include, without limitation: topical, forexample, epicutaneous, inhalational, enema, ocular, otic and intranasaldelivery; enteral, for example, orally, by gastric feeding tube andrectally; and parenteral, such as, intravenous, intraarterial,intramuscular, intracardiac, subcutaneous, intraosseous, intradermal,intrathecal, intraperitoneal, transdermal, iontophoretic, transmucosal,epidural and intravitreal, with oral, intravenous, intramuscular andtransdermal approaches being preferred in many instances.

The compounds may be compounded or otherwise manufactured into asuitable composition for use, such as a pharmaceutical dosage form ordrug product in which the compound is an active ingredient. Compositionsmay comprise a pharmaceutically acceptable carrier, or excipient. Anexcipient is an inactive substance used as a carrier for the activeingredients of a medication. Although “inactive,” excipients mayfacilitate and aid in increasing the delivery or bioavailability of anactive ingredient in a drug product. Non-limiting examples of usefulexcipients include: antiadherents, binders, rheology modifiers,coatings, disintegrants, emulsifiers, oils, buffers, salts, acids,bases, fillers, diluents, solvents, flavors, colorants, glidants,lubricants, preservatives, antioxidants, sorbents, vitamins, sweeteners,etc., as are available in the pharmaceutical/compounding arts.

Useful dosage forms include: intravenous, intramuscular, orintraperitoneal solutions, oral tablets or liquids, topical ointments orcreams and transdermal devices (e.g., patches). In one embodiment, thecompound is a sterile solution comprising the active ingredient (drug,or compound), and a solvent, such as water, saline, lactated Ringer'ssolution, or phosphate-buffered saline (PBS). Additional excipients,such as polyethylene glycol, emulsifiers, salts and buffers may beincluded in the solution.

In one embodiment, the dosage form is a transdermal device, or “patch”The general structure of a transdermal patch is broadly known in thepharmaceutical arts. A typical patch includes, without limitation: adelivery reservoir for containing and delivering a drug product to asubject, an occlusive backing to which the reservoir as attached on aproximal side (toward the intended subject's skin) of the backing andextending beyond, typically completely surrounding the reservoir, and anadhesive on the proximal side of the backing, surrounding the reservoir,typically completely, for adhering the patch to the skin of a patient.The reservoir typically comprises a matrix formed from a non-woven(e.g., a gauze) or a hydrogel, such as a polyvinylpyrrolidone (PVP) orpolyvinyl acetate (PVA), as are broadly known. The reservoir typicallycomprises the active ingredient absorbed into or adsorbed onto thereservoir matrix, and skin permeation enhancers. The choice ofpermeation enhancers typically depends on empirical studies. As is shownin Example 12, below, certain formulations that may be useful aspermeation enhancers include, without limitation: DMSO; 95% PropyleneGlycol+5% Linoleic Acid; and 50% EtOH+40% HSO+5% Propylene Glycol+5%Brij30.

Examples 1-7 are excerpts from United Stated patent application Ser. No.11/565,779, and are recited herein to provide non-limiting illustrationsof useful synthetic methods and efficacies of certainmitochondria-targeting free-radical scavenging compounds utilizingGramicidin S-derived mitochondria-targeting groups.

Example 1

Materials.

All chemicals were from Sigma-Aldrich (St Louis, Mo.) unless otherwisenoted. Heparin, ketamine HCl and sodium pentobarbital were from AbbottLaboratories (North Chicago, Ill.). Dulbecco's modified Eagle medium(“DMEM”) was from BioWhittaker (Walkersville, Md.). Fetal bovine serum(FBS; <0.05 endotoxin units/ml) was from Hyclone (Logan, Utah).Pyrogen-free sterile normal saline solution was from Baxter (Deerfield,Ill.).

General.

All moisture-sensitive reactions were performed using syringe-septum captechniques under an N2 atmosphere and all glassware was dried in an ovenat 150° C. for 2 h prior to use. Reactions carried out at −78° C.employed a CO₂-acetone bath. Tetrahydrofuran (THF) was distilled oversodium/benzophenone ketyl; CH₂Cl₂ (DCM), toluene and Et₃N were distilledfrom CaH₂. Me₂Zn was purchased from Aldrich Company.

Reactions were monitored by thin layer chromatography (“TLC”) analysis(EM Science pre-coated silica gel 60 F254 plates, 250 μm layerthickness) and visualization was accomplished with a 254 nm UV light andby staining with a Vaughn's reagent (4.8 g (NH₄)₆Mo7O₂₄ 4H₂O, 0.2 gCe(SO₄)₂.4H₂O in 10 mL cone. H₂SO₄ and 90 mL H₂O). Flash chromatographyon SiO₂ was used to purify the crude reaction mixtures.

Melting points were determined using a Laboratory Devices MeI-Temp II.Infrared spectra were determined on a Nicolet Avatar 360 FT-IRspectrometer. Mass spectra were obtained on a Waters Autospec doublefocusing mass spectrometer (“EI”) or a Waters Q-Tof mass spectrometer(“ESI”). LC-MS data were obtained on an Agilent 1100 instrument, using aWaters Xterra MS CH 3.5 μm RP column (4.6×100 mm).

Synthesis Example 1

Prepared as a colorless oil (FIG. 3, compound 1) according to theliterature procedure, see Edmonds, M. K. et al. Design and Synthesis ofa Conformationally Restricted Trans Peptide Isostere Based on theBioactive Conformations of Saquinavir and Nelfinavir, J Org Chem.66:3747 (2001); see also Wipf P. et al., Org Lett. 7:103 (2005); seealso Xiao, J. et al., Electrostatic versus Steric Effects inPeptidomimicry: Synthesis and Secondary Structure Analysis of GramicidinS Analogues with (E)-Alkene Peptide Isosteres, J Am Chem Soc. 127:5742(2005).

A solution of 2.20 g (5.52 mmol) of compound 1 (FIG. 3) in 20.0 mL ofdry CH₂Cl₂ was treated at room temperature with 1.85 g (7.17 mmol) ofCp₂ZrHCl. The reaction mixture was stirred at room temperature for 5min, CH₂Cl₂ was removed in vacuo and 20.0 mL of toluene was added. Theresulting yellow solution was cooled to −78° C. and treated over aperiod of 30 min with 2.76 mL (5.52 mmol) of Me-Zn (2.0 M solution intoluene). The solution was stirred at −78° C. for 30 min, warmed to 0°C. over a period of 5 min and treated in one portion with 2.05 g (11.1mmol) of N-Boc-isovaleraldimine, see Edmonds, M. K. et al. J Org Chem.66:3747 (2001); see also Wipf, P. et al., J Org Lett. 7:103 (2005); seealso Xiao et al., J Am Chem Soc. 127:5742 (2005).

The resulting mixture was stirred at 0° C. for 2 h, quenched withsaturated NH₄Cl, diluted with EtOAc, filtered through a thin pad ofCelite, and extracted with EtOAc. The organic layer was dried (MgSO₄),concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,hexane/EtOAc) to yield 3.13 g (97%) as a colorless, oily 1:1 mixture ofdiastereomers.

A solution of 4.19 g (7.15 mmol) of product in 100 mL of drytetrahydrofuran (‘THF’) was treated at 0° C. with 9.30 ml. (9.30 mmol)of tetrabutylammoniumfluoride (TBAF, 1.0 M solution in THF). Thereaction mixture was stirred at room temperature for 20 h. diluted withEtOAc, and washed with brine. The organic layer was dried (MgSO₄),concentrated in vacuo, and purified by chromatography on SiO₂ (4:1,hexane/EtOAc) to yield 1.89 g (76%) as a light yellowish, foamy 1:1mixture of diastereomers.

A solution of 1.86 g (5.23 mmol) of product in 40.0 mL of dry CH₂Cl₂ wastreated at 0° C. with 1.46 mL (10.5 mmol) of triethylamine (“TEA”), 2.02mL (21.4 mmol) of Ac₂O, and 63.9 mg (0.523 mmol) of4-N,N¹-(dimethylamino) pyridine (“DMAP”). The reaction mixture wasstirred at 0° C. for 15 min and at room temperature for 3 h, dilutedwith EtOAc, and washed with brine. The organic layer was dried (MgSO₄),concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,hexane/Et₂O) to yield 1.97 g (94%) of acetic acid(2S)-benzyl-(5R)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester(807 mg, 38.7%), acetic acid(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester(826 mg, 39.6%), and a mixture of the aforementioned species (337 mg,16.2%).

A solution of 350 mg (0.899 mmol) of acetic acid(2S)-benzyl-(5S)-tert-butoxycarbonylamino-7-methyloct-(3E)-enyl ester in8.00 mL of MeOH was treated at 0° C. with 62.0 mg (0.449 mmol) of K₂CO₃.The reaction mixture was stirred at 0° C. for 1 h and at roomtemperature for an additional 4 h, diluted with EtOAc, and ashed withH₂O. The organic layer was dried (MgSO₄), concentrated in vacuo, andpurified by chromatography on SiO₂ (4:1, hexane/EtOAc) to yield 312 mg(quant.) of compound 2 (FIG. 3) as a colorless oil.

A solution of 23.0 mg (66.2 μmol) of compound 2 (FIG. 3) in 2.00 mL ofdry CH₂Cl₂ was treated at 0° C. with 42.1 mg (99.3 μmol) of Dess-MartinPeriodinane. The reaction mixture was stirred at 0° C. for 1 h and atroom temperature for an additional 4 h, quenched with saturated Na₂S₂O₃in a saturated NaHCO₃ solution, stirred for 30 min at room temperature,and extracted with CH₂Cl₂. The organic layer was dried (Na₂SO₄),concentrated in vacuo to give a colorless foam and subsequentlydissolved in 3.00 mL of THE, and treated at 0° C. with 300 μL (600 μmol)of 2-methyl-2-butene (2.0 M solution in THF) followed by anothersolution of 18.0 mg (199 μmol) of NaClO₂ and 18.2 mg (132 μmol) ofNaH₂PO₄.H₂O in 3.00 mL of H₂O. The reaction mixture was stirred at 0° C.for 1 h and at room temperature for an additional 3 h, extracted withEtOAc, and washed with H₂O. The organic layer was dried (Na₂SO₄) andconcentrated in vacuo to yield compound 3 (FIG. 3) as a crude colorlessfoam that was used for the next step without purification.

A solution of crude compound 3 (FIG. 3) (66.2 μmol) in 3.00 mL of CHCl₃was treated at 0° C. with 10.7 mg (79.2 μmol) of 1-hydroxybenzotrizole(“HOBt”) and 14.0 mg (73.0 μmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (“EDC”), followed by a solution of 62.9 mg(132 μmol) of H-Pro-Val-Orn(Cbz)-OMc, see Edmonds, M. K. et al. J OrgChem. 66:3747 (2001); see also Wipf P. et al., J Org Lett. 7:103 (2005),see also Xiao, J. et al., J Am Chem Soc. 127:5742 (2005), in 1.00 mL ofCHCl₃ and 0.8 mg (6.6 μmol) of DMAP. The reaction mixture was stirred atroom temperature for 2 d, diluted with CHCl₃, and washed with H₂O. Theorganic layer was dried (Na₂SO₄), concentrated in vacuo, and purified bychromatography on SiO₂ (from 2:1, hexanes/EtOAc to 20:1, CHCl₃/MeOH) toyield 51.3 mg (94%) of compound 4a (FIG. 3) as a colorless foam.

A solution of 53.7 mg (65.5 μmol) of compound 4a (FIG. 3) in 2.00 mL ofMeOH was treated at 0° C. with 655 μL (655 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 6 h, and treatedwith 655 μL (655 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 5.00mL of CHCl₃ and treated at room temperature with 10.6 mg (78.4 μmol) ofHOBt, 15.1 mg (78.8 nmol) of EDC, 20.2 mg (118 μmol) of 4-amino-TEMPOand 8.0 mg (65.5 μmol) of DMAP. The reaction mixture was stirred at roomtemperature for 36 h, diluted with CHCl₃, and washed with H₂O. Theorganic layer was dried (Na₂SO₄), concentrated in vacuo, and purified bychromatography on SiO₂ (from 1:1, hexane/EtOAc to 20:1, CHCl₃/MeOH) toyield 62.0 mg (99%) of compound 5a (FIG. 3) as a colorless solid. Thefollowing characterization data were obtained: LC-MS (Rt 8.81 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=959.5[M+H]⁺, 981.5 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₅₃H₈₀N₇O₉Na(M+Na) 981.5915, found 981.5956.

A solution of 60.0 mg (71.7 μmol) of compound 4b (FIG. 3), see Tamaki,M. et al. I. Bull Chem Soc Jpn., 66:3113 (1993), in 2.15 mL of MeOH wastreated at room temperature with 717 tL (717 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 5 h, and treated at0° C. with 717 μL (717 μmol) of 1 N HCl. The solution was extracted withCHCl₃ and the organic layer was dried (Na₂SO₄) and concentrated in vacuoto give the crude acid as colorless foam. The acid was dissolved in 6.04mL of CHCl₃ and treated at room temperature with 11.6 mg (85.8 μmol) ofHOBt, 16.5 mg (85.1 μmol) of EDC, 18.5 mg (108 μmol) of 4-amino-TEMPOand 8.8 mg (72.0 μmol) of DMAP. The reaction mixture was stirred at roomtemperature for 20 h, diluted with CHCl₃, and washed with H₂O. Theorganic layer was dried (Na₂SO₄), concentrated in vacuo, and purified bychromatography on SiO₂ (from 2:1, hexane/EtOAc; to 20:1, CHCl₃/MeOH) toyield 69.6 mg (99%) of compound 5b (FIG. 3) as a yellowish solid. Thefollowing characterization data were obtained: LC-MS (Rt 7.02 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=976.5[M+H], 998.4 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₅₂H₇₉N₈O₁₀Na(M+Na) 998.5817, found 998.5774.

A solution of crude compound 3 (FIG. 3) (40.3 μmol) in 3.00 mL of CH₂Cl₂was treated at 0° C. with 10.4 mg (60.7 μmol) of 4-amino-TEMPO, 7.7 mg(40.2 μmol) of EDC, and 5.4 mg (44.2 μmol) of DMAP. The reaction mixturewas stirred at room temperature for 20 h, diluted with CHCl₃, and washedwith H₂O. The organic layer was dried (Na₂SO₄), concentrated in vacuo,and purified by chromatography on SiO₂ (from 4:1 to 1:1, hexane/EtOAc)to yield 18.8 mg (91%) of compound 5c (FIG. 3) as a yellowish solid. Thefollowing characterization data were obtained: LC-MS (Rt 7.01 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=537.3[M+Na]⁺) and HRMS (ESI) in/z calculated for C30H48N3O4Na (M+Na)537.3543, found 537.3509.

Determination of Intracellular Superoxide Radicals.

Oxidation-dependent fluorogenic dye, dihydroethidium (“DHE”, MolecularProbes) was used to evaluate intracellular production of superoxideradicals. DHE is cell permeable and, in the presence of superoxide, isoxidized to fluorescent ethidium which intercalates into DNA. Thefluorescence of ethidium was measured using a FACscan (Becton-Dickinson,Rutherford, N.J.) flow cytometer, equipped with a 488-nm argon ion laserand supplied with the Cell Quest software. Mean fluorescence intensityfrom 10,000 cells were acquired using a 585-nm bandpass filter (FL-2channel).

Determination of Intracellular ATP Levels.

Cells were incubated with 10 μm of compound 5a (FIG. 3) for indicatedperiods of time (2, 4, 6, 12, and 14 h). At the end of incubation, cellswere collected and the content of intracellular ATP was determined usinga bioluminescent somatic cell assay kit (Sigma, St. Louis, Mass.). As apositive control, cells were incubated with 2 mM of 2-dexy-glucose, aglucose analogue which competitively inhibits cellular uptake andutilization of glucose, for 12 and 14 h.

Cells.

Caco-2BBe human enterocyte-like epithelial cells were obtained from theAmerican Type Culture Collection (Manassas, Va.). Cells were routinelymaintained at 37° C. in under a humidified atmosphere containing 8% CO2in air. The culture medium was DMEM supplemented with 10% PBS,non-essential amino acids supplement (Sigma-Aldrich catalogue # M7145),sodium pyruvate (2 mM), streptomycin (0.1 mg/ml), penicillin G (100U/ml) and human transferrin (0.01 mg/ml). The culture medium was changed3 times per week.

Surgical Procedures to Obtain Vascular Access.

All study protocols using rats followed the guidelines for the use ofexperimental animals of the US National Institutes of Health and wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Pittsburgh.

Male specific pathogen-free Sprague Dawley rats (Charles RiverLaboratories, Wilmington, Mass.), weighing 150-250 g, were housed in atemperature-controlled environment with a 12-h light/dark cycle. Therats had free access to food and water. For experiments, rats wereanesthetized with intramuscular ketamine HCl (30 mg/kg) andintraperitoneal sodium pentobarbital (35 mg/kg). Animals were kept in asupine position during the experiments. Lidocaine (0.5 ml of a 0.5%solution) was injected subcutaneously to provide local anesthesia atsurgical cut-down sites. In order to secure the airway, a tracheotomywas performed and polyethylene tubing (PE 240; Becton Dickinson, Sparks,Md.) was introduced into the trachea. Animals were allowed to breathespontaneously.

The right femoral artery was cannulated with polyethylene tubing (PE10). This catheter was attached to a pressure transducer that allowedinstantaneous measurement of mean arterial pressure (MAP) during theexperiment. For experiments using the pressure-controlled hemorrhagicshock (HS) model, the right jugular vein was exposed, ligated distally,and cannulated with polyethylene tubing (PE 10) in order to withdrawblood. For experiments using the volume-controlled hemorrhagic shock(HS) model, the jugular catheter was used to infuse the resuscitationsolution and the right femoral vein, which was cannulated with a siliconcatheter (Chronic-Cath, Norfolk Medical, Skokie, Ill.), was used towithdraw blood.

All animals were instrumented within 30 min. Heparin (500 U/kg) wasadministered immediately after instrumentation through the femoral vein.Animals were placed in a thermal blanket to maintain their bodytemperature at 37° C. The positioning of the different devicesaforementioned was checked postmortem.

Intestinal Mucosal Permeability Assay.

Animals were allowed access to water but not food for 24 h prior to theexperiment in order to decrease the volume of intestinal contents. Therats were instrumented as described above. A midline laparotomy wasperformed and the small intestine was exteriorized from theduodenojejunal junction to the ileocecal valve. A small incision wasmade on the antimesenteric aspect of the proximal small intestine andsaline solution (1.5 ml) was injected. The bowel was ligated proximallyand distally to the incision with 4-0 silk (Look, Reading, Pa.).

The small intestine was compressed gently in aboral direction along itslength to displace intestinal contents into the colon. Starting 5 cmfrom the ileocecal valve, the ileum was partitioned into six contiguouswater-tight segments. Each segment was 3 cm long and was boundedproximally and distally by constricting circumferential 4-0 silksutures. Care was taken to ensure that the vascular supply to intestinewas not compromised, and each segment was well-perfused.

Two randomly selected segments in each rat were injected with 0.3 ml ofvehicle and served as “no treatment” controls. In order to fill thesegments, a small incision was made and the solution was injected usinga Teflon catheter (Abbocath 16Ga, Abbot Laboratories).

The remaining four other segments were injected with solutionscontaining either 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl(TEMPOL) or one of the Gramicidin S-based compounds. Four differentfinal concentrations of TEMPOL in normal saline were evaluated: 0.1, 1,5 and 20 mM. The hemigramicidin-based compounds were dissolved in amixture of dimethylsulfoxide (DMSO) and normal saline (1:99 v/v) andinjected at final concentrations of 0.1, 1, 10 or 100 μM.

After the segments were loaded with saline or the test compounds, thebowel was replaced inside the peritoneal cavity and the abdominalincision was temporarily closed using Backhaus forceps.

After a 5 min stabilization period, hemorrhagic shock was induced bywithdrawing blood via the jugular catheter. MAP was maintained at 30±3mm Hg for 2 hours. The shed blood was re-infused as needed to maintainMAP within the desired range.

After 2 h of shock, the animals were euthanized with an intracardiac KClbolus injection. The ileum was rapidly excised from the ileocecal valveto the most proximal gut segment. The tips of each segment werediscarded. In order to assay caspases 3 and 7 activity and phospholipidsperoxidation, mucosa samples were collected from gut segmentsimmediately after hemorrhage and stored at −80° C. For permeabilitymeasurements, each segment was convened into an everted gut sac, aspreviously described by Wattanasirichaigoon et al., seeWattanasirichaigoon, S. et al. Effect of mesenteric ischemia andreperfusion or hemorrhagic shock on intestinal mucosal permeability andATP content in rats, Shock. 12:127-133 (1999).

Briefly, as per the Wattanasirichaigoon protocol referenced above, thesacs were prepared in ice-cold modified Krebs-Henseleit bicarbonatebuffer (“KHBB”), pH 7.4. One end of the gut segment was ligated with a4-0 silk suture; the segment was then everted onto a thin plastic rod.The resulting gut sac was mounted on a Teflon catheter (Abbocath 16GA,Abbot Laboratories) connected to a 3 ml plastic syringe containing 1.5ml of KHBB. The sac was suspended in a beaker containing KHBB plusfluorescein-isothiocyanate labeled dextran (average molecular mass 4kDa, FD4; 0.1 mg/ml). This solution was maintained at 37° C., andoxygenated by bubbling with a gas mixture (O₂ 95%/CO₂ 5%). After 30 min,the fluid within the gut sac was collected. The samples were cleared bycentrifugation at 2000 g for 5 min.

Fluorescence of FD4 in the solution inside the beaker and within eachgut sac was measured using a fluorescence spectrophotometer (LS-50,Perkin-Elmer, Palo Alto, Calif.) at an excitation wavelength of 492 nmand an emission wavelength of 515 nm. Mucosal permeability was expressedas a clearance normalized by the length of the gut sac with units ofnL·min⁻¹·cm², as previously described, see Yang, R. et al., Ethylpyruvate modulates inflammatory gene expression in mice subjected tohemorrhagic shock, Am J Physiol Gastrointest Liver Physiol 283:G212-G22(2002).

Results for a specific experimental condition (i.e., specific testcompound at a single concentration) were expressed as relative change inpermeability calculated according to this equation: Relative change inpermeability (%)=(C_(Hs exp)−C_(normal))/C_(Hs cont)−C_(normal))×100,where C_(HS exp) is the clearance of FD4 measured for a gut segmentloaded with the experimental compound, C_(normal) is the clearance ofFD4 measured in 6 gut segments from 3 normal animals not subjected tohemorrhagic shock, and C_(HS cont) is the mean clearance of FD4 measuredin 2 gut segments filled with vehicle from the same animal used tomeasure C_(HS exp).

Measurement of Permeability of Caco-2 Monolayers.

Caco-2_(BBe) cells were plated at a density of 5×10⁴ cells/well onpermeable filters (0.4 μm pore size) in 12-well bicameral chambers(Transwell, Costar, Corning, N.Y.). After 21 to 24 days, paracellularpermeability was determined by measuring the apical-to-basolateralclearance of FD4.

Briefly, the medium on the basolateral side was replaced with controlmedium or medium containing menadione (50 μM final). Medium containingFIX (25 mg/ml) was applied to the apical chamber. In some cases, one ofthe gramicidin S-based compounds, XJB-5-131, also was added to theapical side at final concentrations of 0.1, 1, 10 or 100 μM. After 6hours of incubation, the medium was aspirated from both compartments.Permeability of the monolayers was expressed as a clearance(pL·h⁻¹·cm⁻²), see Han, X. et al., Proinflammatory cytokines cause NOdependent and independent changes in expression and localization oftight junction proteins in intestinal epithelial cells, Shock.19:229-237 (2003).

Caspases 3 and 7 Activity Assay.

Caspases 3 and 7 activity was measured using a commercially availableassay kit, Caspase GIo™ 3/7 assay kit (Promega, Madison, Wis.). Briefly,50 μl of rat gut mucosa homogenate (20 jug protein) was mixed with 50 μlof Caspase-GIo™ reagent and incubated at room temperature for 1 hour. Atthe end of incubation period, the luminescence of each sample wasmeasured using a plate reading chemiluminometer (ML1000, DynatechLaboratories, Horsham, Pa.). Activity of caspases 3 and 7 was expressedas luminescence intensity (arbitrary units per mg protein). Proteinconcentrations were determined using the BioRad assay (Bio-RadLaboratories, Inc., Hercules, Calif.).

Assay for Peroxidation of Phospholipids.

Gut mucosal samples were homogenized. Lipids were extracted fromhomogenates using the Folch procedure, see M. Lees and G. H.Sloan-Stanley, A simple method for isolation and purification of totallipids from animal tissue. J. BIOL. CHEM. 226:497-509 (1957), andresolved by 2D HPTLC (High Performance Thin Layer Chromatography) aspreviously described, see Kagan, V. E. et al., A role for oxidativestress in apoptosis: Oxidation and externalization of phosphatidylserineis required for macrophage clearance of cell undergoing Fas-mediatedapoptosis, J Immunol. 169:487-489 (2002). Spots of phospholipids werescraped from HPTLC plates and phospholipids were extracted from silica.Lipid phosphorus was determined by a micro-method, see Bottcher, C. J.F. et al., A rapid and sensitive sub-micro phosphorus determination.Anal Chim Acta. 24: 203-204 (1961).

Oxidized phospholipids were hydrolyzed by pancreatic phospholipase A2 (2U/μl) in 25 mM phosphate buffer containing 1 mM CaCl₃, 0.5 mM EDTA and0.5 mM sodium dodecyl sulfate (SDS) (pH 8.0, at room temperature for 30min). Fatty acid hydroperoxides formed were determined by fluorescenceHPLC of resorufin stoichiometrically formed during their microperoxidase11-catalized reduction in presence of Amplex Red (for 40 min at 4° C.)(8). Fluorescence HPLC (Eclipse XDB-C18 column, 5 pin, 150×4.6 mm,mobile phase was composed of 25 mM disodium phosphate buffer (pH7.0)/methanol (60:40 v/v); excitation wavelength 560 nm, emissionwavelength 590 nm) was performed on a Shimadzu LC-100AT HPLC systemequipped with fluorescence detector (RF-10Axl) and autosampler(SIL-10AD).

Survival of Rats Subjected to Volume-Controlled Hemorrhagic Shock.

Following surgical preparation and a 5-min stabilization period toobtain baseline readings, rats were subjected to hemorrhagic shock.Bleeding was carried out in 2 phases.

Initially, 21 ml/kg of blood was withdrawn over 20 min. Immediatelythereafter, an additional 12.5 ml/kg of blood was withdrawn over 40 min.Thus, hemorrhage occurred over a total period of 60 min and the totalblood loss was 33.5 ml/kg or approximately 55% of the total bloodvolume. Rats were randomly assigned to receive XJB-5-131 (2 μmol/kg) orits vehicle, a 33:67 (v/v) mixture of DMSO and normal saline. XJB-5-131solution or vehicle alone was administered as a continuous infusionduring the last 20 min of the hemorrhage period. The total volume offluid infused was 2.8 ml/kg and it was administered intravenously usinga syringe pump (KD100, KD Scientific, New Hope, Pa.). Rats were observedfor 6 hours or until expiration (defined by apnea for >1 min). At theend of the 6 hour observation period, animals that were still alive wereeuthanized with an overdose of KCl.

Blood pressure was recorded continuously using a commercial strain-gaugetransducer, amplifier, and monitor (S90603a, SpaceLabs, Redmond, Wash.).Blood samples (0.5 ml) were collected from the jugular vein at thebeginning of hemorrhage (baseline), at the end of hemorrhage (shock) andat the end of resuscitation (resuscitation). Hemoglobin concentration[Hb], lactate and glucose concentration were determined using anauto-analyzer (Model ABL 725, Radiometer Copenhagen, Westlake, Ohio).

Data Presentation and Statistics.

All variables are presented as means+Standard Error Mean (SEM).Statistical significance of differences among groups was determinedusing ANOVA (analysis of variance) and LSD (Least SignificantDifference) tests, or Kruskal-Wallis and Mann-Whitney tests asappropriate. Survival data were analyzed using the log-rank testSignificance was declared for p values less than 0.05.

Example 2

Selective delivery of TEMPO to mitochondria could lead totherapeutically beneficial reduction of ROS; therefore, investigation ofthe use of conjugates of 4-amino-TEMPO (“4-AT”) was explored. In orderto selective target the mitochondria, a targeting sequence using themembrane active antibiotic Gramicidin S (“GS”) as well as correspondingalkene isosteres, shown in FIGS. 2 and 3. Accordingly, using theGramicidin S peptidyl fragments and alkene isosteres as “anchors,” theTEMPO “payload” could be guided into the mitochondria.

The Leu-^(D)Phe-Pro-Val-Orn fragment of hemigramicidin was used as atargeting sequence. Alkene Isosteres such as (E)-alkene isosteres ofGramicidin S (i.e., hemigramicidin) were used as part of the targetingsequence. See FIG. 3 for the synthetic pathway for (E)-alkene isosteresand compound 3 for the corresponding chemical structure. The (E)-alkeneas depicted in compound 2 of FIG. 3 was then oxidized in a multi-stepprocess to yield the compound as depicted in compound 3 an example ofthe (E)-alkene isostere.

Then, the compound depicted as compound 3 of FIG. 3 was conjugated withthe tripeptide H-Pro-Val-Orn(Cbz)-OMe using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”) asa coupling agent. The tripeptide is an example of a suitable targetingsequence having affinity for the mitochondria of a cell. The resultingproduct is shown as compound 4a in FIG. 3. Saponification of compound 4afollowed by coupling with 4-amino-TEMPO (“4-AT”) afforded the resultingconjugates shown as compound 5a in FIG. 3, in which the Leu-^(D)Phepeptide bond has been replaced with an (E)-alkene.

In an alternate embodiment, conjugates 5b and 5c in FIG. 3 by couplingthe peptide 4b (Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe) and the (E)-alkeneisostere as indicated as compound 3 in FIG. 3 to 4-AT. The peptide isanother example of a suitable targeting sequence having an affinity withthe mitochondria of a cell.

Electron paramagnetic resonance (“EPR”) spectroscopy was used to monitorthe cellular delivery of compounds 5a and 5b shown in FIG. 3 in mouseembryonic cells (“MEC”).

The following conditions were used during the EPR-based analysis of theintegration and reduction of nitroxide Gramicidin S-peptidyl conjugatesin MECs. The MECs at a concentration of 10 million MECs per ml, wereincubated with 10 μM of 4-AT and compound 5a, respectively. Recoverednitroxide radicals in whole cells, mitochondria, and cytosol fractionswere resuspended in phosphate buffer saline (“PBS”) in the presence andabsence, respectively, of 2 μM K₃Fe(CN)₆. In brief. FIG. 4A shows arepresentative EPR spectra of compound 5a in different fractions of MECsin the presence of K₃Fe(CN)₆. Further, FIG. 4B shows an assessment ofintegrated nitroxides.

Distinctive characteristic triplet signals of nitroxide radicals weredetected in MECs incubated with 10 μM of compound 5a (FIG. 3) as well asin mitochondria isolated from these cells. The cytosolic function didnot elicit EPR signals of nitroxide radicals; similar results wereobserved with conjugate 5b (FIG. 3) (data not shown).

Incubation of MECs with compound 5a (FIG. 3) resulted in integration andone-electron reduction of compound 5a, as evidenced by a significantincrease in magnitude of the EPR signal intensity upon addition of aone-electron oxidant, ferricyanide (FIG. 4B) (Note: EPR results forincubation of MECs with 5b are not shown in FIG. 4; however, EPR resultsfor 5b were similar when compared to 5a). In contrast to 5a and 5b,however, 4-amino-TEMPO (4-AT) did not effectively permeate cells or themitochondria, as shown by the absence of significant amplitude change inthe EPR results for 4-AT.

The ability of 5a, 5b (FIG. 3), and 4-AT to prevent intracellularsuperoxide generation by flow cytometric monitoring of oxidation ofdihydroethidium (“DUE”) to a fluorescent ethidium was tested. Theability of 5a, 5b, and 4-AT to protect cells against apoptosis triggeredby actinomycin D (“ActD”) was also tested. MECs were pretreated with 10μM 4-AT, 5a, or 5b then incubated with ActD at a concentration of 100ng/mL. It was found that 5a and 5b completely inhibited nearly two-foldintracellular superoxide generation in MECs (sec FIG. 6A). 4-AT had noeffect on the superoxide production in MECs.

Apoptotic cell responses were documented using three biomarkers: (1)externalization of phosphatidylserine (“PS”) on the cell surface (byflow cytometry using an FITC-labeled PS-binding protein, annex in V, seeFIGS. 6B and 6E); (2) activation of caspase-3 by cleavage of the7-DEVD-AMC substrate (sec FIG. 6C), and, (3) DNA fragmentation by flowcytometry of propidiium iodide stained DNA (see FIG. 6D).

Phosphatidylserine (“PS”) is an acidic phospholipid located exclusivelyon the inner leaflet of the plasma membrane; exposure of PS on the cellsurface is characteristic of cell apoptosis. Externalization of PS wasanalyzed by flow cytometry using an annexin V kit. Cells were harvestedby trypsinization at the end of incubation and then stained with annexinV-FITC and propidium iodide (“PS”). Ten thousand cell events werecollected on a FACScan flow cytometer. Annexin V-positive andPI-negative cells were considered apoptotic.

Activation of capase-3, a cysteine protease only activated in theexecution phase of apoptosis, was determined using an EnzChek capsase-3assay kit.

Further, calcium and magnesium dependent nucleases are activated thatdegrade DNA during apoptosis. These DNA fragments are eluted, stainedwith propidium iodide and analyzed using flow cytometry. A cellpopulation with decreased DNA content was considered a fraction ofapoptotic cells.

Anti-apoptotic effects of compounds 5a and 5b were observed atrelatively low concentrations of 10 μM. Compounds 5a and 5b (FIG. 3)reduced the number of annexin V-positive cells as shown in FIG. 6B,prevented caspase-3 activation as shown in FIG. 6C, and prevented DNAfragmentation as shown in FIG. 6D. At concentrations in excess of 10 μM,both 5a and 5b were either less protective or exhibited cytotoxicity(FIG. 6E). In contrast, 4-AT afforded no protection.

In contrast, compound 5c, which docs not have a complete targetingmoiety, was ineffective in protecting MECs against ActD-inducedapoptosis (FIGS. 6B and 6C) at low concentrations. Accordingly, thehemigramicidin peptidyl targeting sequence is essential foranti-apoptotic activity of nitroxide conjugates such as those containingTEMPO.

Finally, the reduction of compounds 5a and 5b could also causeinhibition of mitochondrial oxidative phosphorylation, so the ATP levelsof MECs treated with these compounds were tested. As is known to oneordinarily skilled in the art, ATP serves as the primary energy sourcein biological organisms; reduction of ATP levels would greatly impairnormal cell function. ATP levels in MECs in the presence or absence of5a or 2-deoxyglucose (“2-DG”) were used as a positive control (see FIG.6F). At concentrations at which anti-apoptotic effects were maximal (˜10μM, FIG. 6E), nitroxide conjugates did not cause significant changes inthe cellular ATP level Therefore, synthetic GS-peptidyl conjugatesmigrate into cells and mitochondria where they are reduced withoutaffecting the ability of the mitochondria to produce ATP.

Example 3

In an in vivo assay, the ileum of rats was divided into a series ofwell-vascularized components in a manner akin to links of sausage. Thelumen of each ileal compartment was filled with a 3 μL aliquot of testsolution. Two of the ileal compartments were filled with vehicle alone(i.e., a solution containing at least in part the TEMPO derivative).These two components served as internal controls to account forindividualistic variations in the severity of shock or the response ofthe mucosa to the shock.

Using this assay system, eight compounds were evaluated as shown in FIG.5: TEMPOL (FIG. 5A), one dipeptidic TEMPO analog (FIG. 5B—XJB-5-208), 3hemigramicidin-TEMPO conjugates (FIGS. 5C XJB-5-125, 5E XJB-5-131, and5G XJB-5-197), and 3 hemigramicidin compounds that do not have the TEMPOmoiety (FIGS. 5D—XJB-5-127, 5F—XJB-5-133, and 5H—XJB-5-194).

Hemorrhagic shock in rats leads to marked derangements in intestinalmucosal barrier function—in other words, the mucosal permeability ofshocked intestinal segments was significantly greater than thepermeability of segments from normal rats (52.3+0.5 versus 6.9+0.1nL·min⁻¹·cm⁻², respectively; p<0.01), see Tuomincn, E. K. J.,Phospholipid cytochrome c interaction: evidence for the extended lipidanchorage, J. BIOL. CHEM., 277:8822-8826 (2002); also Wipf P. et al.,Mitochondria targeting of selective electron scavengers: synthesis andbiological analysis of hemigramicidin-TEMPO conjugates, J. AM. CHEM.SOC. 127:12460-12461. Accordingly, mice were subjected to 2 hours ofshock (Mean Arterial Pressure (“MAP”)=30 f 3 mm Hg), the gut segmentswere harvested and mucosal permeability to flouresceinisothiocyanate-dextran (“FD4”) measured ex vivo. Data in FIG. 5 areexpressed as a percentage of the change permeability relative to thatobserved in simultaneously assayed control segments loaded during shockwith normal saline solution.

Accordingly, intraluminal TEMPOL was used as a “positive control” forgut mucosal protection assay. TEMPOL concentrations >1 mM in the gutlumen ameliorated hemorrhagic shock-induced ileal mucosalhyperpermeability (FIG. 5A). Two of the TEMPO conjugates, namelyXJB-5-208 (FIG. 5B) and XJB-5-131 (FIG. 5C), also significantlyameliorated hemorrhagic shock-induced ileal mucosal hyperpermeability.The lowest effective concentration for XJB-5-208 (FIG. 5B) and XJB-5-131(FIG. 5E) was 1 μM; i.e., both of these compounds were ˜1000-fold morepotent than TEMPOL. Two other compounds carrying the TEMPO payload,XJB-5-125 (FIG. 5C) and XJB-5-197 (FIG. 5G) failed to provide protectionagainst gut barrier dysfunction induced by hemorrhage. XJB-5-133 (FIG.5F) has the same (hemigramicidin-based) mitochondrial targeting moietyas XJB-5-131 (FIG. 5E) but lacks the TEMPO payload. It is noteworthy,therefore, that XJB-5-133 (FIG. 5F) did not afford protection from thedevelopment of ileal mucosal hyperpermeability.

Ineffective as well were the two other hemigramicidin-based compoundsthat also lacked the TEMPO payload, XJB-5-127 (FIG. 5D) and XJB-5-194(FIG. 5H). Of the compounds screened, XJB-5-131 (FIG. 5E) appeared to bethe most effective, reducing hemorrhagic shock-induced mucosalhyperpermeability to approximately 60% of the control value.

Based upon the results as reflected in FIGS. 5A-51I, both the TEMPOpayload and the “anchoring” hemigramicidin fragment are requisitemoieties that should be present in order for effective electronscavenging activity by the XJB-5-131 compound. Accordingly, it was foundthat XJB-5-131 ameliorates peroxidation of mitochondrial phospholipids(i.e., ROS activity) in gut mucosa from rats subject to hemorrhagicshock.

In the subsequent series of in vivo studies, the affect of intraluminalXJB-5-131 on hemorrhage-induced peroxidation of phospholipids inintestinal mucosa was examined. Isolated segments of the ileum of ratswere divided into a series of well-vascularized components in a mannerakin to sausage and the lumen of each ileal compartment was filled withthe same volume of test solution containing either vehicle or a 10 μMsolution of XJB-5-131, which was previously indicated to be the mostactive of the hemigramicidin-TEMPO conjugates. In a preferredembodiment, 0.3 mL of test solution filled the lumen of each ilealcompartment.

After two hours of HS, samples of ileal mucosa from the gut sacs filledwith the vehicle and XJB-5-131 were obtained and compared with ilealmucosa of normal MECs. All samples were assayed with caspase 3 orcaspase 7 activity as well as the peroxidation of phosphatidylcholine(“PC”), phosphatidylethanolamine (“PE”), phosphatidylserine (“PS”), andcardiolipin (“CL”), summarized in FIG. 7.

As can be seen in FIGS. 7A-7D, treatment with XJB-5-131 significantlyameliorated hemorrhage-induced peroxidation of CL, the only phospholipidtested found in mitochondria. However, treatment with XJB-5-131 only hada small effect on PE peroxidation and no effect on peroxidation of PCand PS. Based upon these trends, hemorrhagic shock is associated withsubstantial oxidative stress even in the absence of resuscitation.Further, this data also establishes that XJB-5-131 is an effective ROSscavenger as it localizes predominantly in mitochondria and protects CLfrom peroxidation.

Relative to the activity measured in samples from normal animals, theactivity of caspases 3 and 7 was markedly increased in vehicle-treatedmucosal samples from hemorrhaged rats (FIG. 8). However, when the ilealsegments were filled with XJB-5-131 solution instead of its vehicle, thelevel of caspase 3 and 7 activity after hemorrhagic shock wassignificantly decreased. Accordingly, hemorrhagic shock is associatedwith activation of pro-apoptotic pathways in gut mucosal cells.Moreover, the data support the view that this process is significantlyameliorated following mitochondrial treatment with XJB-5-131.

Example 4

In another series of experiments, monolayers of enterocyte-like cells,Caco-2_(BBe), were studied for physiological and pathophysiologicalpurposes for determining intestinal barrier function. Just as with theprior Examples with respect to ROS exposure, the permeability ofCaco-2_(BBe) monolayers increases when the cells are incubated with theROS, hydrogen peroxide, or menadione (a redox-cycling quinine thatpromotes the formation of superoxide anion radicals), see Baker, R. D.et al., Polarized Caco-2 cells. Effect of reactive oxygen metabolites onenterocyte barrier function, DIGESTIVE DIS. SCI. 40:510-518 (1995); alsoBanan, A. et al., Activation of delta-isoform of protein kinase C isrequired for oxidant-induced disruption of both the microtubulecytoskeleton and permeability barrier of intestinal epithelia, J.PHARMACOL. EXP. THER. 303:17-28 (2002).

Due to the results with respect to XJB-5-131 and its amelioration ofhemorrhage-induced CL peroxidation in mucosal cells in vivo (seeaforementioned Examples 1 and 2), a possible treatment using XJB-5-131was investigated to determine if menadione-induced epithelialhyperpermeability could be ameliorated in vitro. Consistent with theprior in vivo observations. Caco-2_(BBe) monolayers were incubated inthe absence and in the presence of menadione, respectively. After 6hours, incubation of Caco-2_(BD)c monolayers with menadione caused amarked increase in the apical-basolateral clearance of FD4 (FIG. 9).Treatment with 10 μM XJB-5-131 provided significant protection againstmenadione-induced hyperpermeability.

Example 5

As reflected by the above in vivo and in vitro studies, XJB-5-131 hadsignificantly beneficial effects on several biochemical andphysiological read-outs. Accordingly, systemic administration ofXJB-5-131 was investigated with respect to whether it would prolongsurvival of patients subjected to profound periods of hemorrhagic shockwith massive blood loss in the absence of standard resuscitation withblood and crystalloid solution. As in the above studies, rats wereutilized as test patients.

A total of sixteen rats were tested in this study. Rats were treatedwith 2.8 ml/kg of vehicle or the same volume of XJB-5-131 solutionduring the final 20 min of the bleeding protocol. The total dose ofXJB-5-131 infused was 2 μmol/kg. Following profound hemorrhagic shockconsistent with the protocol described above for the prior studies,thirteen survived for at least 60 min and received the full dose ofeither XJB-5-131 solution or the vehicle, a 33:67 (v/v) mixture of DMSOand normal saline. As shown in Table 2, blood glucose, lactate andhemoglobin concentrations were similar in both groups at baseline andbefore and immediately after treatment. None of the between-groupdifferences were statistically significant.

TABLE 2 End of first End of second phase of phase of Parameter CompoundBaseline hemorrhage hemorrhage Blood glucose Vehicle 143 ± 5  255 ± 30219 ± 26  concentration XJB-5-131 134 ± 4  228 ± 24 201 ± 38  (mg/dL)Blood lactate Vehicle  1.8 ± 0.4  606 ± 0.8 5.9 ± 3   concentrationXJB-5-131  1.8 ± 0.2  5.7 ± 0.8 5.6 ± 1.2 (mEq/L) Blood Hb Vehicle 12.7± 0.5 11.1 ± 0.3 9.4 ± 0.2 concentration XJB-5-131 12.7 ± 0.3 10.7 ± 0.39.4 ± 0.3 (g/dL)

Shortly after treatment was started, mean arterial pressure (“MAP”)increased slightly in both groups (see FIG. 10A). In both groups, meanarterial pressure (“MAP”) decreased precipitously during the first phaseof the hemorrhage protocol and remained nearly constant at 40 mm Hgduring the beginning of the second phase. Six of the seven animals inthe vehicle-treated (control) group died within one hour of the end ofthe bleeding protocol and all were dead within 125 minutes (FIG. 10B).Rats treated with intravenous XJB-5-131 survived significantly longerthan those treated with the vehicle. Three of the six rats survivedlonger than 3 hours after completion of the hemorrhage protocol; one ratsurvived the whole 6 hour post-bleeding observation period (FIG. 10B).

Accordingly, analysis of the XJB-5-131 studies indicate that exposure ofthe patient to the compound prolongs the period of time that patientscan survive after losing large quantities of blood due to traumaticinjuries or other catastrophes (e.g., rupture of an abdominal aorticaneurysm).

By extending the treatment window before irreversible shock develops,treatment in the field with XJB-5-131 might “buy” enough time to allowtransport of more badly injured patients to locations where definitivecare, including control of bleeding and resuscitation with bloodproducts and non-sanguineous fluids, can be provided. The results usinga rodent model of hemorrhagic shock also open up the possibility thatdrugs like XJB-5-131 might be beneficial in other conditions associatedwith marked tissue hypoperfusion, such as stroke and myocardialinfarction.

The results presented here also support the general concept thatmitochondrial targeting of ROS scavengers is a reasonable therapeuticstrategy. Although previous studies have shown that treatment withTEMPOL is beneficial in rodent HS situations, a relatively large dose ofthe compound was required (30 mg/kg bolus+30 mg/kg per h). In contrast,treatment with a dose of XJB-5-131 that was about 300 fold smaller (˜0.1mg/kg) was clearly beneficial. The greater potency of XJB-5-131 ascompared to TEMPOL presumably reflects the tendency of XJB-5-131 tolocalize in mitochondrial membranes, a key embodiment of the invention.As indicated above, two hemigramicidin-4-amino-TEMPO conjugates (namelyXJB-5-208 and XJB-5-131, see FIG. 2) are concentrated in themitochondria of cultures mouse embryonic cells following incubation withsolutions of the compounds.

Further, the use of XJB-5-131 significantly prolonged the survival ofthe rats subjected to massive blood loss, even though the animals werenot resuscitated with either blood or other non-sanguineous fluids andthey remained profoundly hypotensive.

In light of the above, synthetic hemigramicidin peptidyl-TEMPOconjugates permeate through the cell membrane and also the mitochondrialmembrane where they act as free radical scavengers for ROS such as, butnot limited to, superoxide anion radicals. The conjugates are thenreduced within the mitochondria by electron-transport proteins which areinvolved with the cellular respiration pathway, thereby coupling thedecoupled ROS species. These conjugates also have the advantage, asdiscussed above, of being anti-apoptotic, especially in the case ofcompounds such as 5a and 5b.

By effectively reducing the amount of ROS species, a patient'scondition, including an illness or other medical condition, may beameliorated and, in some cases, survival may be prolonged as describedin the Example IV study. Examples of such conditions, including diseasesand other medical conditions, include (but are not limited to) thefollowing medical conditions which include diseases and conditions:myocardial ischemia and reperfusion (e.g., after angioplasty andstenting for management of unstable angina or myocardial infarction),solid organ (lung liver, kidney, pancreas, intestine, heart)transplantation, hemorrhagic shock, septic shock, stroke, tissue damagedue to ionizing radiation, lung injury, acute respiratory distresssyndrome (ARDS), necrotizing pancreatitis, and necrotizingenterocolitis.

Example 6

In a further embodiment, in support of the inter-changeability ofcargoes of the mitochondria-targeting groups, a composition forscavenging radicals in a mitochondrial membrane comprises a radicalscavenging agent or an NOS inhibitor and a membrane active peptidylfragment having a high affinity with the mitochondrial membrane. Themembrane active peptidyl fragment preferably has a property selectedfrom the group consisting of antioxidant, radioprotective, protective,anti-apoptotic, therapeutic, ameliorative, NOS antagonist andcombinations thereof. In a related embodiment, with respect to compoundswith antibiotic properties, it is generally preferable to employcompounds whose mode of action includes bacterial wall targets.

In another embodiment, the membrane active compound is preferablyselected from the group consisting of bacitracins, gramicidins,valinomycins, enniatins, alamethicins, beauvericin, serratomolide,sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinumpeptides.

In a related embodiment, the NOS antagonist is selected from the groupconsisting of XJB-5-234 (a), XJB-5-133 (b), XJB-5-241 (c), and XJB-5-127(d), comprising AMT NOS antagonist cargos:

Example 7

The following examples provide protocols for additional cargo usable incompounds described herein which serve as NOS antagonists.

Compound (1) is Boc-Leu-ψ[(E)-C(CH₃)═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-AMT(XJB-5-241) and was prepared according to the following protocol. Asolution of 11.0 mg (13.2 μmol) ofBoc-leu-ψ[(E)-C(CH₃)═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-OMc (248) in 400 μL ofMeOH was treated at 0° C. with 132 μL (132 nmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 8 h. and treatedwith 132 μL (132 nmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 2.00mL of CHCl₃ and treated at room temperature with 2.1 mg (16 nmol) ofHOBt, 3.0 mg (16 nmol) of EDC, 3.3 mg (20 nmol) of2-amino-5,6-dihydro-6-methy 1-4H-1,3-thiazine.HCl and 3.5 mg (27 nmol)of DMAP. The reaction mixture was stirred at room temperature for 48 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 11 mg (89%)of XJB-5-241 as a colorless powder. The following characterization datawere obtained: LC-MS(R_(t) 8.37 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min. 0.4 mL/min; m/z=932.4 [M+H]⁺) 954.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₅₀H₇₄N₇O₈S (M+H) 932.5320, found 932.5318.

Compound (2) is Boc-leu-ψ[(E)-CH═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-AMT(XJB-5-133) and was prepared according to the following protocol. Asolution of 20.0 mg (24.3 nmol) of 2-85 (XJB-5-194) in 800 nL of MeOHwas treated at 0° C. with 243 μL (243 nmol) of 1 N NaOH. The reactionmixture was stirred at room temperature for 6 h, and treated with 243 μL(243 μmol) of 1 N HCl. The solution was extracted with CHCl₃ and theorganic layer was dried (Na₂SO₄) and concentrated in vacuo to give thecrude acid as a colorless form. This acid was dissolved in 1.00 mL ofCHCl₃ and treated at room temperature with 3.9 mg (29 nmol) of HOBt, 5.6mg (29 μmol) of EDC, 6.1 mg (37 nmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 7.4 mg (61 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 20 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) and an additionalpreparative Cm reverse phase HPLC purification was performed: 80% to100% CH₃CN (H₂O) in 20 min, 5.0 mL/mm) to afford 12.9 mg (58%) ofXJB-5-133 as a colorless powder. The following characterization datawere obtained: LC-MS (R_(t) 7.89 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=918.3 [M+H]⁺, 940.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₄₉H₇₂N₇O₈S (M+H) 918.5163, found 918.5185.

Compound (3) is Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-AMT (XJB-5-127) and wasprepared according to the following protocol. A solution of 24.0 mg(28.7 μmol) of Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe in 800 μL of MeOHwas treated at room temperature with 287 μL (287 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 5 h. and treated at0° C. with 287 μL (287 μmol) of 1 N HCl. The solution was extracted withCHCl₃ and the organic layer was dried (Na₂SO₄) and concentrated in vacuoto give the crude acid as a colorless foam. The crude acid was dissolvedin 2.00 mL of CHCl₃ and treated at room temperature with 4.6 mg (34μmol) of HOBt, 6.6 mg (34 μmol) of EDC, 5.7 mg (34 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 8.8 mg (72.0 μmol)of DMAP. The reaction mixture was stirred at room temperature for 24 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(2:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 17.0 mg (63%)of XJB-5-127 as a colorless powder. The following characterization datawere obtained: LC-MS (R_(t) 6.32 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=935.3 [M+H]⁺, 957.3 [M+Na]⁺) and HRMS(ESI) m/z calculated for C₄₈H₇₁N₈O₉S (M+H) 935.5065, found 935.5044.

Compound (4) is Boc-Leu-ω[(E)-CH═CH]^(D)Phe-AMT (XJB-5-234) A solutionof erode Boc-Leu-ψ[(E)-CH═CH]-^(D)Phe-OH (2-84) (30.5 μmol) in 2.00 mLof CH₂Cl₂ was treated at 0° C. with 6.1 mg (37 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl, 7.0 mg (37 μmol) ofEDC, 4.9 mg (37 μmol) of HOBt, and 9.3 mg (76 μmol) of DMAP. Thereaction mixture was stirred at room temperature overnight, concentratedin vacuo, and purified by chromatography on SiO₂ (2:1, CH₂Cl₂/EtOAc) toyield 9.1 mg (63%) of XJB-5-234 as a colorless foam. The followingcharacterization data were obtained: LC-MS (R_(t) 8.42 min, lineargradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=474.5 [M+H]⁺)and HRMS (ESI) m/z calculated for C₂₆H₄₀N₃O₃S (M+H) 474.2790, found474.2781.

Compound (5) is Boc-Leu-ψ[(Z)—(CF═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-TEMPO(XJB-7-53). A solution of 3.4 mg (4.1 μmol) ofBoc-Leu-ψ[(Z)—CF═CH]-^(D)Phe-Pro-Val-Orn(Cbz)-OMe XJB-5-66) in 400 μL ofMeOH was treated at 0° C. with 41 μL (41 μmol) of 1 N NaOH. The reactionmixture was stirred at room temperature for 12 h, and treated with 41 μL(41 μmol) of 1 N HCl. The solution was extracted with CHCl₃ and theorganic layer was dried (Na₂SO₄) and concentrated in vacuo to give thecrude acid as a colorless form. This acid was dissolved in 400 μL ofCHCl₃ and treated at room temperature will) 0.7 mg (5 μmol) of HOBt, 0.9mg (5 μmol) of EDC, 0.5 mg (4 μmol) of 4-amino-TEMPO and 1.1 mg (6 μmol)of DMAP. The reaction mixture was stirred at room temperature for 12 h,diluted with CHCl₃, and washed with H₂O. The organic layer was dried(Na₂SO₄), concentrated in vacuo, and purified by chromatography on SiO₂(1:1, hexanes/EtOAc followed by 20:1, CHCl₃/MeOH) to yield 3.6 mg (91%)of XJB-7-53 as a colorless powder. The following characterization datawere obtained: LC-MS (R_(t) 8.45 min, linear gradient 70% to 95% CH₃CN(H₂O) in 10 min, 0.4 mL/min; m/z=977.5 [M+H]⁺, 999.5 [M+Na]⁺) and HRMS

(ESI) m/z calculated for C₅₃H₇₉FN₇O₉Na (M+Na) 999.5821.

Compound (6) is Boc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-Orn(Cbz)-Leu-AMT(XJB-7-42) and was prepared according to the following protocol. Asolution of 4.5 mg (5.6 μmol) ofBoc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-Orn(Cbz)-Leu-OMe (2-119) in 0.35 mLof MeOH was treated at 0° C. with 56 μL (56 μmol) of 1 N NaOH. Thereaction mixture was stirred at room temperature for 12 h, and treatedwith 56 μL (56 μmol) of 1 N HCl. The solution was extracted with CHCl₃and the organic layer was dried (Na₂SO₄) and concentrated in vacuo togive the crude acid as a colorless form. This acid was dissolved in 0.80mL of CHCl₃ and treated at room temperature with 0.9 mg (6.7 μmol) ofHOBt, 1.3 mg (6.7 μmol) of EDC, 1.4 mg (8.4 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 1.7 mg (14 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 36 h,concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,CHCl₃/MeOH) to yield 5.0 mg (99%) of XJB-7-42 as a colorless foam. Thefollowing characterization data were obtained: LC-MS (R_(t) 6.61 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=907.3[M+H]⁺, 929.4 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₄₈H₇₂N₇O₈S(M+H) 906.5163, found 906.5190.

Compound (7) is Boc-^(D)Phe-ψ[(E)-C(CH₃)═CH]-Ala-Val-AMT (XJB-7-43). Asolution of 14.3 μmol of crude Boc-^(D)Phe-v[(E)-C(CH₃)═CH]-Ala-Val-OMe(2-111) in 1.00 mL of CHCl₃ was treated at room temperature with 2.3 mg(17 μmol) of HOBt, 3.3 mg (17 μmol) of EDC, 3.6 mg (22 μmol) of2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine.HCl and 4.4 mg (36 μmol) ofDMAP. The reaction mixture was stirred at room temperature for 36 h,concentrated in vacuo, and purified by chromatography on SiO₂ (20:1,CHCl₃/MeOH) to yield 7.5 mg (96%) of XJB-7-43 as a colorless foam. Thefollowing characterization data were obtained: LC-MS (R_(t) 5.41 min,linear gradient 70% to 95% CH₃CN (H₂O) in 10 min, 0.4 mL/min; m/z=545.3[M+H]⁺, 567.3 [M+Na]⁺) and HRMS (ESI) m/z calculated for C₂₉H₄₄N₄O₄S(M+Na) 567.2981, found 567.2971.

Among the preferred radical scavenging agents are a material selectedfrom the group consisting of a ubiquinone analog, a ubiquinone analogfragment moiety, a ubiquinone analog fragment moiety lacking ahydrophilic tail, a superoxide dismutase mimetic, a superoxide dismutasebiomimetic or a salen-manganese compound.

As is known to one ordinarily skilled in the art, ionizing radiationactivates a mitochondrial nitric oxide synthase (“mtNOS”), leading toinhibition of the respiratory chain, generation of excess superoxideradicals, peroxynitrite production and nitrosativc damage. The damagedone by ionizing radiation is believed to be alleviated [See Kanai, A.J. et al., Am J Physiol. 383: F1304-F1312 (2002); and Kanai, A. J. etal. Am J Physiol. 286: H13-H21 (2004)]. The composition of thisembodiment is characterized by the property of inhibiting mtNOS, therebyresisting generation of excess superoxide radicals, peroxynitrite andnitrosative damage.

Protection again irradiation damage using systemic drug delivery canresult in unwanted side effects. One approach to limit or prevent theseadverse side effects is to target drug delivery to the mitochondriausing a peptide carrier strategy.

In one embodiment, a potent NOS inhibitor, the non-arginine analog of2-amino-6-methyl-thiazine (“AMT”), was selected as a cargo. Irradiationof the ureopithelium results in increased production of superoxide andnitric oxide (“NO”), mouse bladders were instilled with AMT or4-amino-TEMPO to determine if inhibition of N O or scavenging freeradicals is more radioprotective.

An unconjugated and conjugated NOS antagonist, (AMT, 100 μM) and anunconjugated and conjugated nitroxide derivative (4-amino-TEMPO, 100 μM)were incubated for two hours at 37° C. with 32D c13 hemopoietic cells.

Following incubation, the cells were lysed and the mitochondria isolatedfor a mass spectrometry analysis where compounds isolated frommitochondria were identified as Na+ adducts. The resulting spectra (notshown) demonstrate that 4-amino-TEMPO only permeate the mitochondrialmembrane with the assistance of the attached GS-derived targetingsequence. Further spectra (not shown) indicate that unconjugated AMT donot enter the mitochondria membrane in substantial quantities. Thus, thetargeting peptides successfully direct a NOS antagonist and a nitroxideto the mitochondria.

Further physiological studies were conducted to determine the effects ofpeptide-targeted AMT and 4-amino-TEMPO on NO and peroxynitriteproduction in irradiated uroepithelial cells. The cells were cultured inan 8-well slide chamber for 3 days and then microsensor measurementswere taken 24 hours after irradiation.

In untreated irradiated cells and cells treated with unconjugated4-amino-TEMPO (100 μM) or unconjugated AMT (10 μM), capsaicin evoked NOproduction and resulted in the formation of comparable amount ofperoxynitrite. In cells treated with high-dose conjugated 4-amin-TEMPO(100 μM), peroxynitrite production was decreased by approximately4-fold. In non-radiated cells or cells treated with conjugated AMT (10μM), NO induced peroxynitrite formation was nearly completely inhibited.This suggested that peptides conjugates couple or covalently link withmembrane impermeant 4-amino-TEMPO or AMT and facilitate the transport of4-amin-TEMPO across the mitochondrial membrane. Furthermore, this datasuggests that the peptide conjugates do not interfere with the NOSinhibitory activity of AMT or the free radical scavenging activity of4-amino-TEMPO and that AMT is a more effective radioprotectant [Kanai,A. J. et al., Mitochondrial Targeting of Radioprotectants Using PeptidylConjugates, ORGANIC AND BIOMOLECULAR CHEMISTRY (in press)].

Quantitative mass spectrometry studies were used to compare theeffectiveness of several AMT peptide conjugates in permeating themitochondrial membrane, specifically XJB-5-234, XJB-5-133, XJB-5-241,and XJB-5-127. The fmole/10 μM mitochondrial protein ratio provides arelative quantification of conjugate concentration at the target site.Table 3 indicates that the most efficacious conjugate was compoundXJB-5-241.

TABLE 3 Compound fmole/10 μM mitochondrial protein XJB-5-234 1.45XJB-5-133 89.8 XJB-5-241 103.3 XJB-5-127 50.8

The trisubstituted (E)-alkene moiety embedded in XJB-5-241 has astronger conformational effect that the less biologically activedisubstituted (b)-alkene XJB-5-133 or the GS peptidyl fragmentXJB-5-127, see Wipf P. et al., Methyl- and (Trifuoromethyl)alkenePeptide Isosteres: Synthesis and Evaluation of Their Potential as β-TurnPromoters and Peptide Mimetics J ORG. CHEM. 63:6088-6089 (1998); alsoWipf, P. et al., Imine Additions of Internal Alkynes for the Synthesisof Trisubstituted (E)-Alkene and Cyclopropane Peptide Isosteres ADV.SYNTH. CAT. 347:1605-1613 (2005). The data indicates that a definedsecondary structure and an appropriate conformational preorganization isimportant in accomplishing mitochondrial permeation of compounds thatreduce nitrosative and oxidative effects.

The presence of a non-hydrolyzable alkene isostere functions in place oflabile peptide bonds and is significant for a prolonged mechanism ofaction. The relatively rigid (E)-alkenes (ψ[(E)-C(R)═CH]) representuseful, conformationally preorganized structural mimetics and have beenused as surrogates of hydrolytically labile amide bonds in a number ofenzyme inhibitors. The primary objective of this strategy is theaccurate mimicry of the geometry of the peptide bond; however,(E)-alkenes also modulate the physicochemical properties, solubility,and lipophilicity, number of hydrogen donors and acceptors, etc, of theparent structures, and therefore generally have a different metabolicfate than simple peptides.

A targeted delivery strategy employed in this invention is advantageoussince some neuronal NOS (nNOS) antagonists and most antioxidants,including nitroxide derivatives, are poorly cell-permeable and requiretherapeutically effective concentrations greater than 100 μM if usedwithout a conjugate.

The method related to this embodiment of the invention delivers acomposition to mitochondria comprising transporting to said mitochondriaa desired cargo which may, for example, be (a) a radical scavengingagent by use of a membrane active peptidyl fragment preferably havinghas a β-turn motif having a high affinity for the mitochondrial membraneor (b) a nitric oxide synthase antagonist bonded to the membrane activepeptidyl fragment.

Example 8—Synthesis of JP4-039 (See FIG. 11)

Synthesis of JP4-039 was accomplished according to the following.

(R,E)-2-Methyl-N-(3-methylbutylidene)propane-2-sulfinamide (1)

(Staas, D. D.; Savage, K. L; Homnick, C. F.; Tsou, N.; Ball, R. G. J.Org. Cham., 2002, 67, 8276)—To a solution of isovaleraldehyde(3-Methylbutyraldehyde, 5.41 mL, 48.5 mmol) in CH₂Cl₂ (250 mL) was added(R)-2-methylpropane-2-sulfinamide (5.00 g, 40.4 mmol), MgSO₄ (5.0 eq,24.3 g, 202 mmol) and PPTS (10 mol %, 1.05 g, 4.04 mmol) and theresulting suspension was stirred at RT (room temperature, approximately25° C.) for 24 h. The reaction was filtered through a pad of Celite® andthe crude residue was purified by chromatography on SiO₂ (3:7,EtOAc:hexanes) to yield 6.75 g (88%) as a colorless oil. ¹H NMR δ 8.07(t, 1H, J=5.2 Hz), 2.47-2.38 (m, 2H), 2.18-1.90 (m, 1H), 1.21 (s, 9H),1.00 (d, 6H, J=6.7 Hz). As an alternative, filtration through a pad ofSiO₂ provides erode imine that functions equally well in subsequentreactions.

(But-3-ynyloxy)(tert-butyl)diphenylsilane (2)

(Nicolaou, K. C. et al., J. Am. Chem. Soc. 2006, 128, 4460)—To asolution of 3-butyn-1-ol (5.00 g, 71.3 mmol) in CH₂Cl₂ (400 mL) wasadded imidazole (5.40 g, 78.5 mmol) and TBDPSCl((tert-butyl)diphenylsilane chloride) (22.0 g, 78.5 mmol) and thereaction was stirred at RT for 22 h. The reaction was filtered through apad a SiO₂, the SiO₂ washed with CH₂Cl₂ and the colorless solutionconcentrated to yield 21.4 g (97%) of crude alkyne that was carried onwithout further purification.

(S,E)-8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-aminehydrochloride (3)

To a solution of (2) (15.9 g, 51.5 mmol) in CH₂Cl₂ (300 mL) was addedzirconocene hydrochloride (15.1 g. 58.4 mmol) in 3 portions and theresulting suspension was stirred at RT for 10 min. The resulting yellowsolution was cooled to 0° C. and Me₃Al (2.0 M in hexanes, 27.5 mL, 54.9mmol) was added and stirred for 5 minutes followed by addition of asolution of imine (1) (6.50 g, 34.3 mmol) in CH₂Cl₂ (50 mL) and theorange solution was stirred for an additional 4 h while allowed to warmto rt. The reaction was quenched with MeOH, diluted with H₂O and CH₂Cl₂and HCl (1 M) was added to break up the emulsion (prolonged stirringwith Rochelle's salt can also be utilized). The organic layer wasseparated and the aqueous layer was washed with CH₂Cl₂ (2×). The organiclayers were combined, washed with brine, dried (MgSO₄), filtered thougha pad of Celite® and concentrated. Since the crude oil was contaminatedwith metal salts, the oil was dissolved in Et2O (diethyl ether,Et=ethyl), allowed to sit for 2 h, and then filtered though a pad ofCelite® and concentrated. Analysis of the crude residue by 1H NMR showedonly 1 diastereomer (>95:5 dr).

To the crude residue in Et₂O (800 mL) was added HCl (4.0 M in dioxane,17.2 mL, 68.7 mmol) and the reaction was stirred for 30 minutes, duringwhich time a white precipitate formed. The precipitate was filtered,washed with dry Et₂O, and dried to afford 11.0 g (74% over 2 steps) of(3) as a colorless solid, mp 151-154° C.; [α]_(D)−2.9 (c 1.0, CH₂Cl₂);¹H NMR δ 8.42 (bs, 3H), 7.70-7.55 (m, 4H), 7.48-7.30 (m, 6H), 5.90 (dt,1H, J=14.9, 7.5 Hz), 5.52 (dd, 1H, J=15.4, 8.4 Hz), 3.69 (appt, 3H,J=6.5 Hz), 2.45-2.20 (m, 2H), 1.80-1.50 (m, 3H), 1.03 (s, 9H), 0.95-0.84(m. 6H); ¹³C NMR δ 135.5, 134.5, 133.7, 129.5, 127.6, 127.3, 63.0, 52.9,42.1, 35.6, 26.7, 24.4, 22.9, 21.5, 19.1; EIMS m/z 395 ([M−HCl]⁺, 40),338 (86), 198 (100); HRMS (EI) m/z calcd for C₂₅H₃₇NOSi (M−HCl)395.2644, found 395.2640.

(S,E)-tert-Butyl8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate (4)

To a solution of (3) (10.5 g, 24.3 mmol) in CH₂Cl₂ (400 mL) was addedEt₃N (triethylamine) (3.0 eq, 10.3 mL, 72.9 mmol) and Boc₂O (1.05 eq,5.74 g, 25.5 mmol) and the resulting suspension was stirred at RT for 14h. The reaction was quenched with sat. aq. NH₄Cl, the organic layersseparated, dried (MgSO₄), filtered and concentrated. The crude residuewas carried onto the next step without further purification.

(S,E)-tert-Butyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (5)

To a solution of crude (4) (12.0 g, 24.3 mmol) in THF (200 mL) at 0° C.was added TBAF (1.0 M in THF, 1.25 eq. 30.4 mL, 30.4 mmol) and thereaction was warmed to RT and stirred for 2 h. The reaction was quenchedwith sat. aq. NH₄Cl, organic layer washed with brine, dried (MgSO₄),filtered and concentrated. The crude residue was purified bychromatography on SiO₂ (3:7, EtOAc:hexanes) to yield 5.51 g (88%, 2steps) as a colorless oil. [α]_(D)−12.7 (c 1.0, CH₂Cl₃); ¹H NMR δ 5.56(dt, 1H, J=15.3, 6.9 Hz). 5.41 (dd, 1H, J=15.4, 6.4 Hz), 4.41 (bs. 1H),4.06 (bm, 1H), 3.65 (appbq, 2H, J=5.7 Hz), 2.29 (q, 2H, J=6.3 Hz), 1.76(bs, 1H), 1.68 (m, 1H), 1.44 (s, 9H), 1.33 (m, 2H), 0.92 (m, 6H); ¹³CNMR δ 155.4, 134.3, 126.9, 79.2, 61.5, 50.9, 44.5, 35.6, 28.3, 24.6,22.5, EIMS m/z 257 ([M]⁺, 10), 227 (55), 171 (65); HRMS (EI) m/z calcdfor C₁₄H₂₇NO₃ 257.1991, found 257.1994.

(S,E)-5-(tert-Butoxycarbonylamino)-7-methyloct-3-enoic acid (6)

To a solution of (5) (1.00 g, 3.89 mmol) in acetone (40 mL) at 0° C. wasadded a freshly prepared solution of Jones Reagent (2.5 M, 3.89 mL, 9.71mmol) and the reaction was stirred at 0° C. for 1 h. The dark solutionwas extracted with Et₂O (3×50 mL), the organic layers washed with water(2×75 mL), brine (1×50 mL), dried (Na₂SO₄), filtered and concentrated toyield 990 mg (94% crude) of acid (6) as a yellow oil that was usedwithout further purification.

TEMPO-4-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (7)

To a solution of (6) (678 mg, 2.50 mmol, crude) in CH₂Cl₂ (35 mL) at 0°C. was added 4-amino tempo (1.5 eq, 662 mg, 3.75 mmol), EDCI (1.2 eq,575 mg, 3.00 mmol), DMAP (1.1 eq, 339 mg, 2.75 mmol) and HOBt-hydrate(1.1 eq, 377 mg, 2.75 mmol) and the resulting orange solution wasstirred at RT for 14 h. The reaction was diluted with CH₂Cl₂, washedwith sat. aq. NH₄Cl and the organic layer dried (Na₂SO₄), filtered andconcentrated. The crude residue was purified by chromatography on SiO₂(1:1 to 2:1, EtOAc/hexanes) to yield 857 mg (76%, 2 steps) as a peachcolored solid, mp 61° C. (softening point: 51° C.); [α]_(D) ²³+35.6 (c0.5. DCM); ESIMS m/z 365 (40), 391 (50), 447 ([M+Na]⁺, 100), 257 (20);HRMS (ESI) m/z calcd for C₂₃H₄₂N₃O₄Na 447.3073, found 447.3109.

The compounds shown as formula 4, above can be synthesized as shown inFIG. 11B. Briefly, synthesis was accomplished as follows: To a solutionof compound (1) in CH₂Cl₂ was added zirconocene hydrochloride, followedby addition of Me₂Zn, then a solution ofN-diphenylphosphoryl-1-phenylmethanimine (Imine). The reaction mixturewas refluxed, filtered, washed, and dried to afford (2). Cleavage of theTBDPS protecting group was achieved by treating (2) with TBAF, whichresulted in the formation of (3). The terminal alcohol (3) wasdehydrated to alkene (4), which was further treated by ozonolysis toafford ester (5). Protocols similar to that given for the synthesis ofJP4-039, above, were used to acylate the amino group with the Bocprotecting group and to react the terminal carboxylic acid with4-amino-TEMPO to afford (6).

Example 9—a Mitochondria-Targeted Nitroxide/Hemigramicidin S ConjugateProtects Mouse Embryonic Cells Against Gamma Irradiation

(see, Jiang, J, et al. Int. J. Radiation Oncology Biol. Phys., Vol. 70,No. 3, pp. 816-825, 2008)

EPR-Based Analysis of Integration and Distribution of Nitroxides.

To compare the integration efficiency, mouse embryonic cells (1×10⁷/mL)were incubated with 10 μM nitroxides for 10 min. ESR spectra ofnitroxide radicals in the incubation medium, cell suspension ormitochondrial suspension were recorded after mixing with acetonitrile(1:1 v/v) after 5-min incubation with 2 mM K₃Fe(CN)₆ using JEOL-RE1X EPRspectrometer under the following conditions: 3350 G center field; 25 Gscan range; 0.79 G field modulation, 20 mW microwave power; 0.1 s timeconstant; 4 min scan time. Integration efficiency was calculated as(E_(initial)−E_(medium))/E_(initial)×100%. Mitochondria were isolatedusing a mitochondria isolation kit (Pierce, Rockford, Ill.) according tothe manufacturer's instruction. Amounts of nitroxide radicals integratedinto mitochondria were normalized to the content of cytochrome c oxidasesubunit IV.

Superoxide Generation.

Oxidation-dependent fluorogenic dye, DHE was used to evaluateintracellular production of superoxide radicals. DHE is cell permeableand, in the presence of superoxide, is oxidized to fluorescent ethidiumwhich intercalates into DNA. Briefly, cells were treated with 5 μM DHEfor 30 min at the end of incubation. Cells were then collected bytrypsinization and resuspended in PBS. The fluorescence of ethidium wasmeasured using a FACScan flow cytometer (Becton-Dickinson, Rutherford,N.J.) supplied with the CellQuest software. Mean fluorescence intensityfrom 10,000 cells was acquired using a 585/42 nm bandpass filter.

CL Oxidation.

CL hydroperoxides were determined by fluorescence HPLC of productsformed in MP-11-catalyzed reaction with a fluorogenic substrate, AmplexRed. Oxidized phospholipids were hydrolyzed by pancreatic phospholipaseA₂ (2 U/ml) in 25 mM phosphate buffer containing 1 mM CaCl₂, 0.5 mM EDTAand 0.5 mM SDS (pH 8.0 at RT for 30 min). After that Amplex Red andMP-11 were added and samples were incubated for 40 min at 4° C. ShimadzuLC-100AT vp HPLC system equipped with fluorescence detector (RF-10Axl,Ex/Em=560/590 nm) and autosampler (SIL-10AD vp) were used for theanalysis of products separated by HPLC (Eclipse XDB-C18 column, 5 μm,150×4.6 mm). Mobile phase was composed of NaH₂PO₄ (25 mM, pH7.0)/methanol (60:40 v/v).

Phosphatidylserine (PS) Externalization.

Externalization of PS was analyzed by flow cytometry using annexin-Vkit. Briefly, harvested cells were stained with annexin-V-FITC and PIfor 5 min in dark prior to flow cytometry analysis. Ten thousand eventswere collected on a FACScan flow cytometer (Becton-Dickinson) suppliedwith CellQuest software.

Gamma-Irradiation Dose Survival Curves of Mouse Embryonic Cells.

Cells were plated in 35-mm Petri dishes with 2 ml culture medium at adensity between 100 and 1000 cells per dish. Cells were treated withGS-nitroxide (XJB-5-125) either before (10-min) or after (1-h)γ-irradiation. XJB-5-125 was removed from the medium 4-hpost-irradiation. Colonies were fixed and stained with 0.25% crystalviolet and 10% formalin (35% v/v) in 80% methanol for 30 min after a9-day incubation period, and those of ≥50 cells were counted assurvivors. The surviving fraction was calculated as the platingefficiency of the samples relative to that of the control.

FIG. 12 shows that nitroxide conjugate XJB-5-125 integrates into cellsand mitochondria much more efficiently than their parent non-conjugated4-amino-TEMPO in mouse embryonic cells. (A) shows their cellular andmitochondrial integration efficiencies in mouse embryonic cells, and (B)shows representative EPR spectrum of nitroxides recovered frommitochondria.

FIG. 13 reveals that nitroxide conjugate XJB-5-125 protects mouseembryonic cells against gamma irradiation induced superoxide generationand cardiolipin peroxidation. (A) superoxide generation. Cells wereexposed to 10 Gy of γ-irradiation. XJB-5-125 (20 μM) was added to cellseither 10-min before or 1-h after irradiation and removed after 5-hincubation. Cells were incubated with 5 μM DHE for 30 min at theindicated time points. Ethidium fluorescence was analyzed using aFACScan flow cytometer supplied with CellQuest software. Meanfluorescence intensity from 10,000 cells was acquired using a 585-nmbandpass filter. (B) Cardiolipin oxidation. Cardiolipin hydroperoxideswere determined using a fluorescent HPLC-based Amplex Red assay. Datapresented are means±S.E. (n=3). *p<0.01 vs non-irradiated cells; *p<0.01(0.05) vs irradiated cells without XJB-5-125 treatment under the samecondition. Insert is a typical 2D-HPTLC profile of phospholipids fromcells.

FIG. 14 reveals that nitroxide conjugate XJB-5-125 protects cellsagainst gamma irradiation induced apoptosis. (A) XJB-5-125 blocksγ-irradiation induced accumulation of cytochrome c in the cytosol ofmouse embryonic cells. (B) Densitometry ratio of cytochrome c/actin.Semi-quantitation of the bands was carried out by densitometry usingLabworks Image Acquisition and Analysis Software (UVP, Upland, Calif.)The level of cytochrome c release was expressed as the mean densitometryratio of cytochrome c over actin. (C) Dose (5, 10 and 20 μM) dependentradioprotective effect of XJB-5-125 (pre-treatment) on γ-irradiation (10Gy) induced phosphatidylserine (PS) externalization. After 48 hpost-irradiation incubation, cells were harvested and stained withannexin-V-FITC and propodium iodide (PI) prior to flow cytometryanalysis. (D) Time (2, 3, 4, 5, and 6 h) dependent radioprotectiveeffect of XJB-5-125 (20 μM) on γ-irradiation (10 Gy) induced PSexternalization (48 h post irradiation) in mouse embryonic cells, (E)Effect of XJB-5-125 on γ-irradiation (10 Gy) induced PS externalizationin human bronchial epithelial cell line BEAS-2B cells. Cells weretreated with 5-125 (5 or 10 μM) before (10-min) or after (1-h)irradiation. Externalization of PS was analyzed 72 h post-irradiationexposure. Data shown are means±S.E. (n=3). *(&)p<0.01(0.05) vsirradiated cells without 5-125 treatment, # p<0.05 vs cells pre-treatedwith 5-125.

FIG. 15 shows the effect of nitroxide conjugate XJB-5-125 ongamma-irradiation dose survival curves of mouse embryonic cells. Cellswere pre- (10-min) or post-treated (1-h) with XJB-5-125 (20 μM), whichwas removed after 4-h incubation period. The surviving fraction wascalculated as the plating efficiency of the samples relative to that ofthe control. The data was fitted to a single-hit multitarget model usingSigmaPlot 9.0 (Systat Software). Data presented are the mean±S.E. (n=3).

FIG. 16 illustrates the effect of GS conjugated nitroxide, XJB-5-125, ongamma-irradiation dose survival curves of 32D cl 3 murine hematopoieticcells. The cells incubated in XJB-5-125 or Tempol had an increased Do(1.138 or 1.209 Gy, respectively) compared to the 32D cl 3 cells (0.797Gy). The cells incubated in XJB-5-125 had an increased shoulder on thesurvival curve with an n of 18.24 compared to 5.82 for the cellsincubated in tempol.

Example 10—Testing of the Radioprotective Abilities of JP4-039

FIGS. 17A and 17B are graphs showing GS-nitroxide compound JP4-039increases survival of mice exposed to 9.75 Gy total body irradiation. InFIG. 17A, mice received intraperitoneal injection of 10 mg per kilogramof each of the chemicals indicated in FIG. 5, then 24 hours laterreceived 9.75 Gy total body irradiation according to published methods.Mice were followed for survival according to IACUC regulations. Therewas a significant increase in survival of mice receiving JP4-039compared to irradiated control mice. (P=0.0008). In FIG. 17B, micereceived intraperitoneal injection of JP4-039 either 10 minutes before(square symbols) or 4 hours after (triangle symbols) irradiation with9.75 Gy.

FIG. 18 is a graph showing that GS-nitroxide compound JP4-039 increasessurvival of mice exposed to 9.5 Gy total body irradiation. Groups of 15mice received intraperitoneal injection of 10 mg. per kilogram of eachindicated GS-nitroxide compound or carrier (Cremphora plus alcohol at 1to 1 ratio, then diluted 1 to 10 in distilled water). Mice received 10mg per kilogram intra-peritoneal injection 24 hours prior to total bodyirradiation. Control mice received radiation alone. There was astatistically significant increase in survival in mice receivingGS-nitroxide compounds. (P=0.0005)

FIG. 19 is a graph showing that GS-nitroxide JP4-039 is an effectivehematopoietic cell radiation mitigator when delivered 24 hr afterirradiation. Irradiation survival curves were performed on cells fromthe 32D cl 3 mouse hematopoietic progenitor cell line, incubated in 10μM JP4-039 for 1 hour before irradiation, or plated in methylcellulosecontaining 10 μM JP4-030 after irradiation. Cells were irradiated from 0to 8 Gy, plated in 0.8% methylcellulose containing media, and incubatedfor 7 days at 37° C. Colonies of greater than 50 cells were counted anddata analyzed by linear quadratic and single-hit, multi-target models.Cells incubated in JP4-039 were more resistant as demonstrated by anincreased shoulder on the survival curve with an ft of 5.25±0.84 if drugwas added before irradiation or 4.55±0.47 if drug was added afterirradiation compared to 1.29+0.13 for 32D cl 3 cells alone (p=0.0109 or0.0022, respectively).

FIG. 20 is a graph showing that JP4-039 is an effective mitigator ofirradiation damage to KM101 human marrow stromal cells. KM101 cells wereincubated in media alone or in JP4-039 (10 μM) for one hour beforeirradiation or 24 hours after irradiation. The cells were irradiated todoses ranging from 0 to 6 Gy and plated in 4 well plates. Seven dayslater the cells were stained with crystal violet and colonies of greaterthan 50 cells counted. Cells incubated in JP4-039 either before or afterirradiation were more radioresistant as shown by an increased shoulderof n=2.3±0.2 or 2.2±0.2, respectively compared to n of 1.1±0.1 for theKM101 cells (p=0.0309 or 0.0386, respectively). There was no significantchange in the Do for the different conditions.

Example 11—NOD/SCID Mouse Model to Optimize JP4-039 for a Clinical Trial

We have significant preliminary data on use of NOD/SCID mice to test theeffects of JP4-039 on human marrow stromal cell and hematopoietic stemcell recovery from total body irradiation to doses that cause thehematopoietic syndrome. FIG. 21A shows results with detection of humancells in NOD/SCID mouse marrow harvested 27 days after cord bloodtransplanted I.V, showing flow cytometric analysis and identification ofhuman CD45+(light gray) hematopoietic cells in NOD/SCID mouse BMfollowing irradiation, proximal tibia bone drilling (see below), andhuman cord blood injection.

-   -   Six NOD/SCID mice were irradiated to 350cGy and injected with        1×10⁷ human cord blood (CB) mononuclear cells (MNC). Five months        after the CB MNC cells were initially injected, the right leg of        6 mice was irradiated to 10 Gy. 24 hours post-irradiation holes        were drilled in the tibiae. (See FIG. 21B) Drill bit size 1 mm.        diameter (Dremel Corp). 24 hours post-bone drilling 1×10⁷ CB MNC        was injected into 3 of the 6 mice. Control mice (3) received no        CB. 27 days after the CB was injected, the bones were harvested        for histochemical analysis and flow cytometric analysis for        human CD45+ cells (light grey) in the BM using a PE-conjugated        anti-CD45 antibody (BD Biosciences). Analysis was performed on a        BD LSR II flow cytometer (BD Biosciences). Human CD45+ cells        were delectable in all of the mice (numbers 1-3) that received        human CB MNC when compared to control mice (mouse 4). The        percent of CD45+ cells ranged from 0.045-3.288 percent in the        non boosted leg and from 0.028-0.892 percent in the high dose        irradiated leg. There was no difference between the        boost-irradiated and non boosted leg in these mice. Although the        data suggest that there is a trend (the percent of human CD45+        cells was lower in the high dose irradiated leg), there was no        statistically significant difference the total body irradiated        non boosted compared to 1000 cGy boosted leg (p=0.25). Day 7        bone photo shown in FIG. 21B.

FIG. 21B is a photomicrograph of cross-section through a tibial wound7-days after surgical construction with a drill bit of a unicortical2-mm diameter wound in the lateral aspect of the tibia 2-mm below theproximal epiphyseal plate. Robust trabecular bone fills theintramedullary canal as well as die cortical window in this intermediatephase of spontaneous wound repair. This time point is optimal forassessing inhibition of marrow stromal cell mediated osteogenesis byirradiation and restoration by JP4-039, as proposed in this application.Arrows indicate margins of the wound. (Toluidine blue stain, ×35)(58)

Example 12—Topical and Transdermal Absorption of GS-Nitroxide

A practical skin patch is planned for delivery of JP4-039 or othercompounds delivered herein. The patch can be administered to a subjectbefore, during or after exposure to radiation, including 24 hr or laterafter irradiation exposure of the subject. In preliminary studies, wesought to characterize the absorption/penetration of a topically appliedrepresentative GS-Nitroxide XJB-5-125 in mouse skin. XJB-5-125 wasselected as a potential topical agent based on its ability to inhibitROS generation, inhibit apoptosis and suppress oxidative damage tomitochondrial lipids. XJB-5-125 comprises the (Leu-D-Phe-Pro-Val-Orn)segment of XJB-5-125 and has been shown to attenuate ActD-induced PSexternalization in a dose-dependent manner of 2.5-20 μM. It can alsoinhibit the release of cytochrome c from mitochondria and suppress CLperoxidation. The physical properties of a chemical are critical to itsability to penetrate into and through the skin. Two important factorsare the log octanal/water (Ko/w) partition coefficient and the molecularweight. For XJB-5-125, the log Ko/w=4.5 and molecular weight is 956. Thelipophilicity “rule” is based on the need for a compound to partitionout of the lipophilic stratum corneum and into the more hydrophilicepidermis and dermis. The log Ko/w and MW of XJB-5-125 are similar toketaconazole (log Ko/w=4.34, MW=532), clotrimazole (log k/ow=6.27,MW=902), and Indomethecin (log Ko/w=4.23, MW=358) suggesting feasibilityof delivery using formulations similar to those used to effectivelydeliver these agents. Like JP4-039, XJB-5-125 is a radiation mitigatoras well as a protector (see FIG. 15).

A small piece of skin (2 cm²) was placed in a Bronaugh styleflow-through diffusion cell system (PermeGear, Riegelsville, Pa.) (FIG.22). It was then sandwiched between two pieces of the inert polymerKel-F and clamped shut to prevent leakage. The epidermal side facesupward and is exposed to the donor solution (test solution), and thedermal side is in contact with the receptor fluid. The exposed surfacearea is 0.79 cm² (circular chamber with 1 cm diameter). The skin forms awater-tight seal in the flow through chamber so the receiving fluid(PBS+25% ethanol) on the dermal side will contain the XJB-5-125 only ifit has penetrated through the skin. The receiver chamber was perfusedwith this buffer that then passes to a fraction collector via Teflontubing. The PBS+25% ethanol was used because it is an effective sink forhydrophobic compounds and produces better in vitrolin vivo correlationsthan other receiver solutions. The skin was maintained at 32° C. byplacing the chamber in a metal block heated via a recirculating waterbath. The skin was equilibrated for 60 minutes prior to introduction ofthe test compound. Seventy five μL of XJB-5-125 was placed on the skinand was allowed to remain for the course of the experiment. The effluxwas collected for 24 hours. (FIGS. 23-25).

To evaluate XJB-5-125 penetration in mouse skin, C57/BL6 mice wereshaved using animal clippers (#40 blade), followed by a brief treatmentwith Nair (depilatory) to remove remaining hair. The skin was washedimmediately after hair removal to prevent further irritation. The skinwas allowed to recover for 24 hours prior to study. This reducesinterference by hair and allows time for small abrasions to heal priorto dermal penetration studies.

Upon completion of the study, the skin was removed from the diffusionchamber. The stratum corneum, which will contain the majority of thetopically applied compound, but is not relevant from a therapeuticstandpoint, was removed by sequential tape-stripping (15 times) usingBrookman Tape (3M, Minneapolis, Minn.). The remaining skin (viableepidermis and dermis) and transdermal effluent were assayed forXJB-5-125 via ESR.

Mouse skin was homogenized in 400 μL 50 mM PBS pH 7.4. EPR measurementswere performed m gas-permeable Teflon tubing (0.8 mm internal diameter,0.013 mm thickness) obtained from Alpha Wire Corp. (Elizabeth, N.J.,USA) on a JEOL JES-REIX spectrometer at 25° C. The Teflon tube(approximately 8 cm in length) was filled with 70 μL of samplecontaining 28.5% of acetonitrile and 2 mM K₃Fe(CN)₆, folded in half, andplaced into an open EPR quartz tube (inner diameter of 3.0 mm). (FIG.24)

EPR spectra were recorded at 334.7 mT, center field; 20 mW, power; 0.079mT, field modulation; 5 mT, sweep width; 400 and 4000, receiver gain;0.1 s, time constraint. Spectra were collected using EPRware software(Scientific Software Services, Bloomington, Ill., USA).

These preliminary experiments demonstrate that XJB-5-125 cansufficiently penetrate intact skin. Farther, the total transdermalabsorption after 24 hours and the level of XJB-5-125 present in theviable skin can be successfully measured using the techniques describedherein. The effect of formulation on topical delivery was examined byusing three different donor solutions (FIG. 25). Donor A=1 mM XJB-5-125in DMSO, donor B=1 mM XJB-5-125 in 95% Propylene Glycol+5% LinoleicAcid, and Donor C=1 mM XJB-5-125 in 50% EtOH+40% HSO+5% PropyleneGlycol+5% Brij30. A total of 75 nmole was placed on top of each piece ofskin to begin these experiments (75 ul of 100 mM). The delivery ofXJB-5-125 into the skin resulted in between 0.07% and 0.46% remainingwithin the skin after 24 hours. The higher delivery rate is in the rangeof other topical products.

Given the observation that XJB-5-125 is active in cells in theconcentration range from 2.5-20 μM and assuming a tissue density of 1g/cm³, an order of magnitude analysis based on these data indicates thatthe topical delivery of XJB-5-125 method to enhance systemic bloodlevels to protect bone marrow is feasible.

Additionally, the fact that the total skin absorption is generallyregarded as linearly related to the donor concentration implies thattopical delivery will be greatly enhanced by increasing the donorconcentration. These preliminary studies demonstrate feasibility ofXJB-5-125 delivery to therapeutic levels and indicate that the smallerJP4-039 molecule, as well as other compounds described herein, may beuseful as a skin patch-deliverable radiation mitigator of thehematopoietic syndrome.

Example 13 (Proposed)

The following can be used to select and optimize the best GS-nitroxideJP4-039 (radiation damage mitigator drug) that can enhance human bonemarrow stromal cell and fresh human stromal cell line seeding efficiencyinto irradiated limbs of NOD/SCID mice. MnSOD-overexpressing cells are apositive control.

Experiments with KM101-MnSOD/ds-red (control KM101-ds-red) clonal celllines. Groups of 12 NOD/SCID mice receive 300 cGy total body irradiation(low dose leg) and a 1000 cGy boost to the left hind leg (high doseleg), then 24 hours later intravenous injection of 1×10⁵ or 1×10⁶ cellsof each cell line (groups 1 and 2). Group 3 is mice that receiveMnSOD-PL intravenously 24 hours prior to irradiation and then injectionof KM101-MnSOD/ds-red. Group 4 is MnSOD-PL intravenously 24 hours priorto irradiation, then control KM101/ds-red cells. This experiment may berepeated twice. Mice will have bone marrow flushed from the hind limbsat days 1, 3, 7, 14 after cell transplantation, and scoring of thepercent of total cells and number of colony forming cells recoverablewhich are ds-red positive thus of human origin. The scoring may be byds-red positivity, and then by colony formation in vitro by stromalcells. We may score the total, then the percent of stromal cells ofhuman origin.

Experiments demonstrating improvement in human bone marrow stromal cellline KM101 seeding by mitochondrial targeted radiationprotection/mitigation JP4-039 (GS-nitroxide) administration. Thisexperiment may be conducted essentially as described above (A), with allgroups, but with a sub-group receiving JP4-039 (24 hours) afterradiation (same day as cell lines are injected, or a sub-group receivingintraperitoneal JP4-039 (daily or weekly after cell linetransplantation). Cells may be explanted from the high dose and low doseirradiated femurs at days 7, 14, 21, and cultured in vitro for humanstromal colony forming progenitor cells (CFU-F). The percent and totalnumber of human cells entering the high dose and low dose irradiatedlimbs can be quantitated by cell sorting for ds-red. Each experiment canbe completed twice.

Experiments as in (A) above, but substituting fresh human marrow Stro1+stromal cells from a 45 y.o. donor.

Experiments as in (B) above substituting Stro1+ human marrow stromalcells.

Statistical Considerations

In (A), we propose comparing at 4 different time points between 4 groupswhere either MnSOD or no MnSOD, and either 10⁵ or 10⁶ KM101 cells areinjected, in terms of the number of DsRed-KM101 cells. In (B), wepropose comparing at 3 different time points between 10 groups wheredifferent doses and schedules of the experimental compound will be used,in terms of the same endpoint as in (A). (C) and (D) are the same as (A)and (B) respectively, except that human stromal cells are used in placeof KM101 cells. All the comparisons in this task are performedseparately for high and low dose radiated legs. ANOVA followed byTukey's test can be used for these analyses. Sample size can beestimated by the two sample t-test for pairwise comparisons. Due to thelack of preliminary data, sample size estimation is based on theexpected difference to detect between groups in terms of the commonstandard deviation σ. Six mice per group can be sacrificed per timepoint. With this sample size, there will be 82% power to detect adifference of 1.8σ between groups using the two sided two sample t testwith significance level 0.05.

As the secondary endpoint, the number of colony forming unit fibroblast(human) CFU-F can also be compared between groups with the same methodas the primary endpoint.

It is expected that MnSOD overexpression in KM101-MnSOD/ds-red cellswill lead to a higher seeding efficiency into both the high and low doseirradiated limbs of NOD/SCID mice. We expect that MnSOD-PL treatment ofthe hematopoietic microenvironment prior to KM101 clonal line cell lineinfusion will further enhance engraftment of both KM101-MnSOD/ds-red andKM101-ds-red cell lines. We expect the highest percent of seedingefficiency will be detected in the mice receiving MnSOD-PL prior toirradiation and injection of KM101-MnSOD/ds-red cells.

We expect that JP4-039 administration daily after cell transplantationwill facilitate improved stability of engraftment of all stromal celllines by decreasing free radical production by the irradiated marrowmicroenvironment.

An inactive control compound for JP4-039 may be used, (JP4-039 absentthe nitroxide active moiety). Based upon the results of theseexperiments, the optimal condition for bone marrow stromal cell seedingcan be derived, and these conditions may be used in experimentsdescribed below.

Example 14 (Proposed)

Selection and optimization of a GS-nitroxide JP4-039 therapy to enhancehuman CD34+ cord blood multilineage hematopoietic stem cell progenitorcell seeding into irradiated limbs of NOD/SCID mice that have beenprepared by engraftment of human marrow stromal cells.

-   -   i. Experiments with TBI treated C57BL/6J mice and mouse marrow        screening, (preliminary system test)    -   ii. Experiments using the optimal seeding protocol for human        KM101 cells into irradiated NOD/SCID mice (anticipated to be        those mice receiving MnSOD-PL prior to irradiation, and then        injection with KM101-MnSOD/ds-red, supplemented with JP4-039        daily). Mice can then receive intravenous injection of 1×10⁵ or        1×10⁵ CD34+ LIN− cells from human umbilical cord blood origin.        Control cells may be CD34+ LIN+(differentiated progenitor) cells        10⁵ or 10⁶ per injection. Groups of 12 mice.

These experiments may be carried out in two schedules.

-   -   Injection of cord blood cells at the same time as        KM101-MnSOD/ds-red cells.    -   Injection of cord blood cells at time of optimal recovery of        KM101-MnSOD/ds-red cells from the explant experiments of        Example 13. This should be at day 7 or day 14 after stromal cell        injection.

In these experiments, mice can be followed and tested at serial timepoints out to two months after cord blood stem cell transplantation. Thepercent of human peripheral blood hematopoietic cells can be scored inweekly peripheral blood samples and number of cells forming CFU-GEMMcolonies can be tested in explanted bones from sacrificed mice.

At days 7, 14, 21, 28, or 60 after cord blood transplantation, mice insub-groups may be sacrificed, and all cells flushed from the high doseand low dose irradiated femurs, and assays carried out for humanmultilineage hematopoietic progenitors-CFU-GEMM. Assays may be carriedout by two methods:

-   -   i. Sorting human CD34+ cells with monoclonal antibodies specific        for human.    -   ii. Colony formation in human CFU-GEMM culture medium and then        secondary scoring of human colonies as the subset of total mouse        and human colony forming cells detected at days 7 and days 14 in        vitro.

In vitro experiments may be carried out in parallel as follows:

KM101-MnSOD-PL plateau phase stromal cells may be irradiated in vitro to100, 200, 500, 1000 cGy, and then CD34+ LIN− human cord blood cellsco-cultivated with the stromal cells in vitro. Controls can includeunirradiated KM101-MnSOD/ds-red, irradiated KM101-ds-red cells,unirradiated KM101-ds-red.

We can score human cobblestone islands (stem cell colonies) on thesecultures weekly, plot cumulative cobblestone island formation,cumulative non-adherent cell production with weekly cell harvest, andassay of weekly cell harvest for CFU-GEMM formation. These studies maybe carried out over two-three weeks. In vitro co-cultivation studies canonly partially duplicate the in vivo hematopoietic microenvironment, andthus two weeks should be the maximum efficient time for detection ofwhether MnSOD-PL expression in the adherent KM101 layer will increaseengraftment of cord blood stem cells.

3. Experiments with JP4-039 supplementation of the cord bloodtransplantation program as in (1) above to increase homing, stablequiescence, and repopulation capacity of human cord blood stem cells byremoving ROS production in the irradiated marrow stromal cellenvironment.

Experiments in vitro supplementing in co-cultivation culture media thedrug JP4-039 daily. The experiments with irradiated KM101 subclonallines, co-cultivated with cord blood stem cells may be carried out withthe addition of JP4-039, or an active analog JP4-039 daily. Controlexperiments can include addition of CD34+ LIN+ differentiated cord bloodcells that are expected to produce fewer CFU-GEMM over time. Stromalcell cultures may be irradiated, cord blood cells added, and culturesscored as above.

Groups of 12 mice can receive the optimal protocol for human CFU-GEMMcell engraftment from the experiment above, and then sub-groups can betreated as follows:

-   -   i. JP4-039 twice weekly.    -   ii. JP4-039 daily.    -   iii. Inactive JP4-039 analog daily.

4. Experiments as in (1) above substituting fresh human Stro1+ marrowcells for KM101 subclonal lines.

5. Experiments as in (2) above substituting human Stro1+ marrow cellsfor KM101 subclonal lines.

Statistical Considerations

In (1), we can compare at 5 different time points between 7 groups wherewe use MnSOD-KM101 and/or 10⁵/10⁶ CD34+ cells, in terms of the number ofCD45+ cells. In (2), we can compare at 5 different lime points between 7groups that use KM101, CD34+ cells, KM101 plus CD34+ cells, theexperimental compound single or double administrations, or inactiveanalog of the experimental compound single or double administrations, interms of the same endpoint as in (1). Tasks (3) and (4) are the same as(A) and (B) of Example 13, respectively, except that we can use humanStro1+ marrow cells in place of KM101 cells. All the comparisons in thistask can be performed separately for high and low dose radiated legs.ANOVA followed by Tukey's test can be used for these analyses. Similarto the sample size considerations in Example 13, we will use 6 mice pergroup at each time point. As the secondary endpoint, the number ofCFU-GEMM can also be compared between groups with the same method as theprimary endpoint.

Likely Outcomes

Based on the results of Example 13, we expect that cord blood stem celland human bone marrow stromal cell homing in vitro will be optimized byMnSOD-PL treatment of the mouse microenvironment prior to stromal celltransplantation, and that MnSOD-PL overexpressing KM101 cells will showfurther stability in the irradiated microenvironment. We expect thatJP4-039 treatment will further enhance hematopoietic cell survival andincrease CFU-GEMM in numbers.

Example 15 (Proposed)

These experiments utilize osteogenesis by human stromal cells as ameasure of effective mitigation of marrow injury by JP4-039. JP4-039 canbe tested for repair of artificial fracture of the proximal tibiae inNOD/SCID mice by human stromal cell derived osteoblasts producing humancollagen and can show enhanced fracture healing by antioxidant JP4-039treatment.

Experiments with mice engrafted with KM101-MnSOD/ds-red compared toKM101 cells. Mice may have holes drilled in both proximal tibias asdescribed above, then irradiation 300 cGy total body dose, 1000 cGy toone hind limb, and then 24 hours later injection of 1×10⁵ bone marrowstromal cells of each line. Mice can be followed for 21 days and atserial seven days time points tibias explanted and assayed for relativecontent of human collagen in the healed bones.

JP4-039 weekly or daily supplemented injections in a repeat experimentof experiment described in (A) (12 mice per group).

Mice receiving MnSOD-PL intravenously 24 hours prior to irradiation (onthe day of bone drilling), and then injection of eitherKM101-MnSOD/ds-red or KM101-ds-red.

Mice receiving scrambled sequence MnSOD-PL injection prior to cell lineinjection as described in (C) above.

) Experiments as in (A-D) substituting fresh Stro1+ stromal cells forKM101 subclones.

Statistical Considerations

In (A), we compare at 3 different time points 17 groups that use KM101cells, MnSOD, the experimental compound single or doubleadministrations, scrambled MnSOD, or a combination of some of these, interms of the percent of human collagen. (B) is the same as (A) exceptthat human Stro1+ marrow cells are used in place of KM101 cells. All thecomparisons in this task can be performed separately for high and lowdose radiated legs. ANOVA followed by Tukey's test can be used for theseanalyses. Similar to the sample size considerations in Example 13, wecan use 6 mice per group at each time point.

Likely Outcomes

We expect that KM101-MnSOD/ds-red will demonstrate improved osteogeniccapacity in vivo. We anticipate that MnSOD-PL administration to mice 24hours prior to irradiation will further enhance homing and osteoblastdifferentiation of KM101-MnSOD/ds-red.

Preliminary data show radiation survival curves of bone marrow stromalcell lines and enhancement of survival by MnSOD overexpression. Otherpreliminary data are expected to show that each Stro1+ cell transfectedwith MnSOD-PL and KM101-MnSOD/ds-red as well as KM101-ds-red are capableto differentiation to osteoblasts in vitro (osteogenic media experimentsin progress) and in vivo in hole drilled NOD/SCID mice. Radiationsurvival curves of KM101-MnSOD/ds-red and KM101-ds-red treated withJP4-039, but not the inactive analog of JP4-039 are shown above. Weanticipate that three conditions: 1) MnSOD-PL administration to themicroenvironment, 2) overexpression of MnSOD in bone marrow stromal celllines of human origin, and 3) supplementation of JP4-039 antioxidanttherapy will lead to maximum osteogenic differentiation by human origincollagen producing cells. As further controls for the experiments, wecan determine whether hematopoietic cells of human origin are requiredfor optimal functioning of bone marrow stromal cells. KM101-MnSOD/ds-redstromal cell seeded NOD/SCID mice can be supplemented with injection ofhuman cord blood CD34+ LIN−, or CD34+ LIN+ cells administered eitherwith the stromal cells, or 24 hours later, to see if these cells produceoptimal colony formation. Other controls can be CD34+ LIN−, CD34+ LIN+hematopoietic cells only. Other controls may include STRO1+ stromalcells progenitors from cord blood alone or whole cord blood controls.

Example 16

To assess the effectiveness of XJB-5-131 in inhibiting degenerationand/or signs of aging, the compound was administered, over an 18-21 weekperiod, progeroid Ercc1^(−/Δ) mice, at a dose of 2 mg/kg in sunfloweroil carrier (to promote solubility) administered intraperitoneally threetimes per week (FIG. 32). Sunflower seed oil was administered to twinErcc1^(−/Δ) mice according to the same schedule as a control. Thetreated and control mice were monitored twice a week for weight andsymptom/sign development.

FIG. 33 presents a summary table showing the results of the treatmentwith XJB-5-131 (“XJB” in this figure), relative to control (sunflowerseed oil) after treatment from 5 wks of life until death. The numbersindicate the average age at onset of each age-related symptom for micetreated with XJB-5-131 or vehicle only (oil) (n=5 mice per group). Cellshighlighted in yellow indicate symptoms that were significantly delayedby XJB-5-131. In addition to improvement in most signs measured, theoverall aging score was significantly improved in the XJB-5-131-treatedmice. Of note, all of the signs of neurodegeneration, includingdystonia, trembling, ataxia, wasting and urinary incontinence weredelayed in the treated animals, providing strong evidence that XJB-5-131protects neurons against degenerative changes caused by oxidativestress.

To assess the ability of XJB-5-131 to inhibit deterioration ofintervertebral discs (an index of degenerative disease of the vertebra),the level of glycosaminoglycan in the discs in treated and control micewere measured, and the results are shown in FIG. 34. The intervertebraldiscs of treated mice contained approximately 30 percent moreglycosaminoglycan relative to control mice, indicating inhibition ofdisc degeneration.

As a measure of the effect of XJB-5-131 on photoaging, Ercc1^(−/cond);K14-Cre mice, which are missing ERCC1 only in the skin, were shaved,treated with a depilatory then irradiated with UV-B light to induce asunburn (500 J/m², the median erythemal dose). Subsequently, the micewere treated with XJB-5-131 (80 μg) emulsified in a cream daily for fivedays. The results, shown in FIG. 35, indicate that the skin of treatedmice appeared much more smooth and healthy relative to control (micetreated with cream only).

At a macroscopic level, administration of XJB-5-131 appears to have beenwell-tolerated by the animals, as indicated by the fact that they didnot lose weight as a result of treatment. Graphs showing weight overtime of treated, untreated and control animals are shown in FIG. 36A-B.To assess the impact of XJB-5-131 at a cellular level a number ofexperiments were carried out using mouse embryonic fibroblasts (“MEF”)cells harvested from Ercc1^(−/−) mouse embryos. As shown in FIG. 37,such MEF cultures were prepared and grown under ambient oxygen(oxidative stress) conditions, and then either untreated (media only) ortreated with a concentration of 500 nM (in media) XJB-5-131, and thentested for SA-β galactosidase staining (a marker of cellularsenescence). The amount of staining was notably less in the treatedcells. In addition, XJB-5-131 treatment was found to reduce the numberof γH2AX foci in DNA (a second marker of cellular senescence as well asDNA double-strand breaks) (FIG. 38), although it did not reduce theamount of apoptosis (FIG. 39).

Example 17—Protective Effects of JP4-039

To assess the therapeutic potential of JP4-039, tests for safety andprotective activity were performed. FIGS. 35 and 36 show the results oftests to evaluate whether varying concentrations of JP4-039 producetoxic effects after 48 hours in cultures of MEF cells prepared fromErcc1^(−/−) or wild-type mouse embryos, respectively. Even under thehighest concentrations tested (10 μM), no signs of toxicity wereobserved in either culture system and cellular proliferation is enhancedrelative to untreated control cells (media only).

To test the protective activity of JP4-039, cultures of primary MEFswere prepared from Ercc1^(−/−) mouse embryos and grown under 20% oxygen(ambient air), which creates oxidative stress in these cells that arehypersensitive to reactive oxygen species. The cells were then eithertreated with a concentration of 1 uM XJB-5-131, JP4-039, JED-E71-37 orJED-E71-58, or left untreated (media), and then after 48 hrs the levelof p16, a marker of irreversible cellular senescence, was measured byimmunofluorescence staining. As seen in FIG. 37, the level of p16 wasmuch lower in MEF cells treated with JP4-039 relative to its level inuntreated cells, whereas XJB-5-131, JED-E71-37 and JED-E71-58 wereobserved to be less effective at this concentration.

Example 18—Protective Effects of Antioxidants in Cell Culture

To evaluate the protective activities of JED-E71-37 and JED-E71-58,primary MEF cells were prepared from Ercc1^(−/−) mice and grown underconditions of oxidative stress (ambient air, 20% oxygen). The cells werethen either untreated or treated with 1 μM JED-E71-37 or JED-E71-58 fora period of 48 hours. As can be seen in FIG. 38, both compounds improvedcell proliferation despite the oxidative stress.

Next, the abilities of these two agents, as well as XJB-5-131 andJP4-039, each at a concentration of 1 μM, were tested for their abilityto prevent oxidation-induced DNA double-strand breaks in cell culturesprepared, and oxidatively stressed, as in the preceding paragraph.Treated as well as untreated cells were, after 48 hours of treatment,immunostained for γ-H2AX, a marker of DNA double-strand breaks as wellas cellular senescence. Results for JED-E71-58 are shown in FIG. 39,showing a distinct decrease in γ-H2AX. JP4-039 was also effective, butXJB-5-131 and JED-E71-37 were observed to be less effective at thisconcentration.

Example 19—Alternative Designs of Nitroxide Analogues

To further investigate the structural requirements for high activity ofGS-nitroxide compound JP4-039, we have designed several nitroxideanalogues. FIG. 40 shows a schematic of alternative designs of nitroxideanalogues. The design can encompass one or both of: modification of thetargeting group to optimize the drug-like properties and/orinvestigation of alternative nitroxide containing groups to improvetheir oxidant efficiency (for example and without limitation, see Reid,D. A. et al. The synthesis of water soluble isoindoline nitroxides and apronitroxide hydroxylamine hydrochloride UV-VIS probe for free radicals.Chem Comm. 1998, 17:1907-8; Iwabuchi, Y. J., Exploration andExploitation of Synthetic Use of Oxoammonium Ions in Alcohol Oxidation.J. Synth. Org. Chem. Jpn. 2008, 66(11):1076-84). Modification of thetargeting group can include replacement of Boc for alternativeprotecting groups, such as Ac (—C(O)CH₃), Cbz (—C(O)O-Bn, where Bn is abenzyl group) or dialkylphosphates. Dialkylphosphates include —P(O)-Ph₂,where Ph is a phenyl group. Other modifications also include isostericreplacement of the alkene group within the targeting group, such as witha cyclopropane group. The nitroxide containing group includes TEMPO andTEMPOL, as well as alternative nitroxide moieties, such as TMIO(1,1,3,3-tetramethylisoindolin-2-yloxyl) or 1-Me-AZADO (1-methyl2-azaadamantane N-oxyl). Synthesis protocols of these alternativenitroxide moieties are provided below.

FIG. 41 shows a synthetic protocol that can be used to produce variousalternative designs of nitroxide analogues, including JP4-039, compoundsaccording to Formula 2, compounds according to Formula 3, and otheranalogues. The specific synthesis of JP4-039 has been described above inExample 8. JP4-039 and its analogues were prepared via an efficientmethod for the asymmetric synthesis of allylic amines, previouslydeveloped in our laboratory (Wipf P. & Pierce J. G. Expedient Synthesisof the α-C-Glycoside Analogue of the Imnmnostimulant Galactosylceramide(KRN7000), Org. Lett. 2006, 8(15):3375-8). One key step in FIG. 41includes use of the zirconium methodology to produce a diastereomericallylic amine (7). This methodology includes hydrozirconation of alkyne(5) with Cp₂ZrHCl, transmetalation to Me₃Al, and addition toAZ-tBu-sulfinyl amine (3). The Smith cyclopropanation of the alkene (8b)with Zn(CH₂I)₂ is another key step in FIG. 41. In tins latter step, thestereochemistry around the cyclopropane ring is to be determined afterthe reaction.

Synthesis of compounds (10a, JP4-039), (10b), (10c), (14a), and (14b)(shown in FIG. 41) was accomplished according to the following.

(R,E)-2-Methyl-N-(3-methylbutylidene)propane-2-sulfinamide (3)

The synthesis of the title compound has already been described inExample 8 (compound 1).

(But-3-ynyloxy)(tert-butyl)diphenylsilane (5)

The synthesis of the title compound has already been described inExample 8 (compound 2).

(S,E)-8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-aminehydrochloride (7)

The synthesis of the title compound has already been described inExample 8 (compound 3).

(S,E)-tert-Butyl8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate (8a)

The synthesis of the title compound has already been described inExample 8 (compound 4).

(S,E)-Benzyl8-(tert-butyldiphenylsilyloxy)-2-methyloct-5-en-4-ylcarbamate (8b)

To a mixture of the amine 7 (1.50 g, 3.79 mmol) in dry THF (15 mL) wereadded Et₃N (1.65 mL, 11.75 mmol), and then a solution of benzylchloroformate (CbzCl, 0.59 mL. 4.17 mmol) in dry THF (4 mL) at 0° C. Theresulting white suspension was allowed to warm to rt and stirred for 5h, then diluted with DCM and water. The aqueous phase was extracted withDCM (2×), and the combined organic layers were washed with 10% HCl andsat. NaHCO₃, dried (MgSO₄), filtered and concentrated in vacuo. Flashchromatography (SiO₂, 8:2, hexanes/EtOAc) afforded 1.45 g (72%) of thetitle compound as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.75-7.65 (m,4H), 7.50-7.28 (m, 11H), 5.70-5.55 (m, 1H), 5.40 (dd, 1H, J=15.4, 6.2Hz), 5.11 (s, 2H), 4.58 (m, 1H), 4.21 (m, 1H), 3.71 (t, 2H, J=6.6 Hz),2.30 (q, 2H, J=6.6 Hz), 1.67 (m, 1H), 1.40-1.22 (m, 2H), 1.07 (s, 9H),0.92 (m, 6H); HRMS (ESI) m/z calcd for C₃₃H₄₃NO₃SiNa 552.2910, found552.2930.

(S,E)-N-(8-(tert-Butyldiphenylsilyloxy)-2-methyloct-5-en-4-yl)-P,P-diphenylphosphinicamide (8c)

To a solution of the amine 7 (400 mg, 1.01 mmol) in dry DCM (7 mL) wereadded Et₃N (0.44 mL, 3.13 mmol), and then a solution ofdiphenylphosphinic chloride (Ph₂POCl, 0.22 mL, 1.11 mmol) in dry DCM (3mL) at 0° C. After being stirred at 0° C. for 15 min, the reactionmixture was allowed to warm to rt and stirred for 4 h, then diluted withDCM and 10% HCl. The aqueous phase was extracted with DCM and thecombined organic layers were washed with sat. NaHCO₃, dried (MgSO₄),filtered and concentrated in vacuo to afford 720 mg of the crude titlecompound as a pale yellow solidified oil, which was used for the nextstep without further purification.

(S,E)-tert-Butyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9a)

The synthesis of the title compound has already been described inExample 8 (compound 5).

(S,E)-Benzyl 8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9b)

To a solution of the TBDPS-protected alcohol 8b (584 mg, 1.10 mmol,crude) in dry THF (9 mL) at 0° C. was added TBAF (1.0M/THF, 1.38 mL,1.38 mmol), and the reaction mixture was allowed to warm to rt whilestirring under argon for 3.5 h, then quenched with sat. aq. NH₄Cl anddiluted with EtOAc. The aqueous phase was separated and extracted withEtOAc. The combined organic layers were washed with brine, dried(Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography(SiO₂, 5:5, hexanes/EtOAc) afforded 194 mg (60%, 2 steps) of the titlecompound as a colorless oil. [α]_(D) ²³ −6.4 (c 1.0, DCM); ¹H NMR (300MHz, CDCl₃) δ 7.20-7.40 (m, 5H), 5.65-5.49 (m, 1H), 5.44 (dd, 1H,7=15.3, 6.6 Hz), 5.09 (s, 2H), 4.67 (bs, 1H), 4.16 (m, 1H), 3.63 (bs,2H), 2.28 (q, 2H, J=6.0 Hz), 1.82 (bs, 1H), 1.65 (m, 1H), 1.40-1.25 (m,2H), 0.80-1.00 (m, 6H); HRMS (ESI) m/z calcd for C₁₇H₂₅NO₃Na 314.1732,found 314.1739.

(S,E)-N-(8-Hydroxy-2-methyloct-5-en-4-yl)-P,P-diphenylphosphinic amide(9c)

To a solution of the TBDPS-protected alcohol 8c (700 mg, 0.983 mmol,crude) in dry THF (8 mL) at 0° C. was added TBAF (1.0M/THF, 1.23 mL,1.23 mmol), and the reaction mixture was allowed to warm to rt whilestirring under argon. As completion was not reached after 4 h, 0.75 eqof TBAF (0.75 mL) was added at 0° C. The reaction mixture was stirredfurther at rt for 3 h, then quenched with sat. aq. NH₄Cl and dilutedwith EtOAc. The aqueous phase was separated and extracted with EtOAc.The combined organic layers were washed with brine, dried (Na₂SO₄),filtered and concentrated in vacuo. Hash chromatography (SiO₂, 95:5,EtOAc/MeOH) afforded 272 mg (77%, 2 steps) of the title compound as awhite solid, mp 124.0-124.2° C.; [α]_(D) ²³ −12.1 (c 1.0, DCM); ¹H NMR(300 MHz, CDCl₃) δ 8.00-7.83 (m, 4H), 7.58-7.35 (m, 6H), 5.52 (dd, 1H,J=15.3, 9.0 Hz), 5.24 (m, 1H), 4.58 (bs, 1H), 3.78-3.47 (m, 3H), 2.80(appdd, 1H, J=9.2, 3.8 Hz), 2.16 (m, 2H), 1.68 (bs, 1H), 1.55-1.43 (m,1H), 1.43-1.31 (m, 1H), 0.87 (dd, 6H, J=8.6, 6.4 Hz); HRMS (ESI) m/zcalcd for C₂₁H₂₈NO₂PNa 380.1755, found 380.1725.

TEMPO-4-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide(10a, JP4-039)

The synthesis of the title compound has already been described inExample 8 (compound 7).

TEMPO-4-yl-(S,E)-5-(benzyloxycarbonylamino)-7-methyloct-3-enamide (10b)

To a solution of the alcohol 9b (158 mg, 0.543 mmol) in acetone (5 mL)at 0° C. was added slowly a freshly prepared solution of Jones reagent(2.5M, 0.54 mL, 1.358 mmol). The resulting dark suspension was stirredat 0° C. for 1 h, then diluted with Et₂O and water. The aqueous phasewas separated and extracted with Et₂O (2×). The combined organic layerswere washed with water (2×) and brine (1×), dried (Na₂SO₄), filtered andconcentrated in vacuo to yield 166 mg (quant.) of the crude acid as aslightly yellow oil, that was used for the next step without furtherpurification.

To a solution of this acid (160 mg, 0.524 mmol, crude) in dry DCM (7 mL)at 0° C. were added successively a solution of 4-amino-TEMPO (139 mg,0.786 mmol) in dry DCM (0.5 mL), DMAP (71 mg, 0.576 mmol), HOBt H₂O (78mg, 0.576 mmol) and EDCI (123 mg, 0.629 mmol). The resulting orangesolution was stirred at rt under argon for 15 h, and then washed withsat. NH₄Cl. The aqueous phase was separated and extracted once with DCM,and the combined organic layers were dried (Na₂SO₄), filtered andconcentrated in vacuo. Hash chromatography (SiO₂, 5:5 to 3:7,hexanes/EtOAc) afforded 171 mg (71%) of the title compound as a peachcolored foam, mp 60.5° C. (softening point: 44° C.); [α]_(D) ²³+26.5 (c0.5, DCM); EIMS m/z 458 ([M]⁺, 37), 281 (19), 154 (28), 124 (47), 91(100), 84 (41); HRMS (EI) m/z calcd for C₂₆H₄₀N₃O₄ 458.3019, found458.3035.

TEMPO-4-yl-(S,E)-5-(diphenylphosphorylamino)-7-methyloct-3-enamide (10c)

To a solution of the alcohol 9c (166.5 mg, 0.466 mmol) in acetone (5 mL)at 0° C. was slowly added a freshly prepared solution of Jones reagent(2.5M, 0.47 mL, 1.165 mmol). The resulting dark suspension was stirredat 0° C. for 2 h, then diluted with Et₂O and water. The aqueous phasewas separated and extracted with Et₂O (2×). The combined organic layerswere washed with water (2×) and brine (1×), dried (Na₂SO₄), filtered andconcentrated in vacuo to yield 114 mg (66%) of the crude acid as a whitefoam, that was used for the next step without further purification.

To a solution of this acid (110 mg, 0.296 mmol, crude) in dry DCM (3.5mL) at 0° C. were added successively a solution of 4-amino-TEMPO (78.4mg, 0.444 mmol) in dry DCM (0.5 mL), DMAP (40.2 mg, 0.326 mmol),HOBt.H₂O (44.0 mg, 0.326 mmol) and EDCI (69.5 mg, 0.355 mmol). Theresulting orange solution was stirred at rt under argon for 13 h, andthen washed with sat. NH₄Cl. The aqueous phase was separated andextracted once with DCM, and the combined organic layers were dried(Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography(SiO₂, EtOAc to 97:3, EtOAc/MeOH) afforded 91.2 mg (59%) of the titlecompound as an orange oil which solidified very slowly upon high vacuum,mp 168.0-168.8° C. (softening point: ˜75° C.); [α]_(D) ²³ −14.1 (c 0.5,DCM); EIMS m/z 525 ([M+H]⁺, 10), 371 (27), 218 (28), 201 (74), 124(100), 91 (35), 84 (26); HRMS (EI) m/z calcd for C₃₀H₄₃N₃O₃P 524.3042,found 524.3040.

Benzyl(1S)-1-(2-(2-(tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-methylbutylcarbamate(11b)

To a solution of ZnEt₂ (110 mg, 0.844 mmol) in dry DCM (2 mL) was addedDME (distilled, 0.088 mL, 844 mmol). The reaction mixture was stirred atrt for 10 min under N₂, then cooled to −20° C. and CH₂I₂ (0.137 mL,1.687 mmol) was added dropwise over 4 min. After stirring for 10 min, asolution of the alkene 8b (149 mg, 0.281 mmol) in dry DCM (1 mL) wasadded dropwise over 5 min. The reaction mixture was allowed to warm tort while stirring. After 10 h, the reaction mixture was quenched withsat. aq. NH₄Cl and diluted with DCM and water, the aqueous phase wasseparated and extracted with EtOAc. The combined organic layers weredried (Na₂SO₄), filtered and concentrated in vacuo. Rash chromatography(SiO₃, 9:1, hexanes/Et₂O) afforded 785 mg (68%) of the title compound asa colorless oil. ¹H NMR analysis showed only 1 diastereomer (>95:5 dr),[α]_(D) ²³ −26.8 (r 1.0, DCM); ¹H NMR (300 MHz, CDCl₃) δ 7.73-7.66 (m,4H), 7.48-7.28 (m. 11H), 5.13-4.96 (m, 2H), 4.62 (appbd, 1H, J=8.4 Hz),3.72 (appbt, 2H, J=6.4 Hz), 3.21 (m, 1H), 1.80-1.63 (m, 1H). 1.60-1.25(m, 4H), 1.08 (s, 9H), 0.92 (appd, 6H, J=6.3 Hz), 0.79 (m, 1H), 0.51 (m,1H), 0.40 (m, 1H), 0.30 (m, 1H); HRMS (ESI) m/z calcd for C₃₄H₄₅NO₃SiNa566.3066, found 566.3103.

(1S)-1-(2-(2-(Tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-methylbutan-1-amine(12)

A flask containing a solution of the Cbz-protected amine 11b (460 mg,0.846 mmol) in a 5:1 MeOH/EtOAc mixture (12 mL) was purged and filled 3limes with argon, then 10% Pd/C (50 mg) was added. The flask was purgedand filled 3 times with H₂, and the resulting black suspension wasstirred at rt under H₂ (1 atm). Since the reaction did not reachcompletion after 3 h, an additional amount of 10% Pd/C (30 mg) was addedand stirring under H₂ was continued for 5 h. The reaction mixture wasthen filtered through a pad of Celite, the Celite washed with MeOH andAcOEt, and the solution concentrated in vacuo to yield 317 mg (92%) ofthe crude title compound as a pale yellow oil, that was used for thenext step without further purification.

Tert-butyl(1S)-1-(2-(2-(tert-butyldiphenylsilyloxy)ethyl)cyclopropyl)-3-methylbutylcarbamate(11a)

To a solution of the amine 12 (309 mg, 0.755 mmol) in dry DCM (12 mL)was added Et₃N (0.21 mL, 0153 mmol) and then Boc₂O (183 mg, 0.830 mmol)at 0° C. The reaction mixture was stirred at rt under N₂ for 28 h. Thereaction was quenched with sat. aq. NH₄Cl and the aqueous phaseextracted with DCM The combined organic layers were dried (Na₂SO₄),filtered and concentrated in vacuo to yield 471 mg of the crude titlecompound as a colorless oil, that was used for the next step withoutfurther purification.

Tert-butyl (1S)-1-(2-(2-hydroxyethyl)cyclopropyl)-3-methylbutylcarbamate(13a)

To a solution of the crude TBDPS-protected alcohol 11a (464 mg, 0.742mmol) in dry THE (6 mL) at 0° C. was added TBAF (1.0M/THE, 0.93 mL,0.927 mmol), and the reaction mixture was allowed to warm to rt whilestirring under N₂. Since TLC showed uncomplete reaction after 5 h. 0.75eq. TBAF (0.56 mL) was added. After 9 h, the reaction mixture wasquenched with sat. aq. NH₄Cl and diluted with EtOAc. The aqueous phasewas separated and extracted with EtOAc. The combined organic layers werewashed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo.Flash chromatography (SiO₁, 5:5, hexanes/EtOAc) afforded 177 mg (88%) ofthe title compound as a colorless oil which solidified upon high vacuumto give a white powder, mp 49.8-50.2° C.; [α]_(D) ²³ −30.8 (c 1.0, DCM),¹H NMR (300 MHz, CDCl₃) δ 4.50 (appbd, 1H, J=4.5 Hz), 3.66 (bs, 2H),2.94 (m, 1H), 2.36 (bs, 1H), 1.82 (bs, 1H), 1.71 (m, 1H), 1.45 (s, 9H),1.39 (t, 2H, J=7.2 Hz), 1.01 (bs, 2H), 0.90 (dd, 6H, J=10.2, 6.6 Hz),0.50 (m, 1H), 0.43-0.27 (m, 2H); HRMS (ESI) m/z calcd for C₁₅H₂₉NO₃Na294.2045, found 294.2064.

Benzyl (1S)-1-(2-(2-hydroxyethyl)cyclopropyl)-3-methylbutylcarbamate(13b)

To a solution of the TBDPS-protected alcohol 11b (320 mg, 0.588 mmol) indry THE (5 mL) at 0° C. was added TBAF (1.0M/THF, 0.74 mL, 0.735 mmol),and the reaction mixture was allowed to warm to rt while stirring underargon for 7 h, then quenched with sat. aq. NH₄Cl and diluted with EtOAc.The aqueous phase was separated and extracted with EtOAc. The combinedorganic layers were washed with brine, dried (Na₂SO₄), filtered andconcentrated in vacuo. Hash chromatography (SiO₂, 5:5, hexanes/EtOAc)afforded 166 mg (92%) of the title compound as a colorless oil. [α]_(D)²³ −21.6 (c 1.0, DCM); ¹H NMR (300 MHz, CDCl₃) δ 7.42-7.28 (m, 5H), 5.10(m, 2H), 4.76 (appbd, 1H, J=5.7 Hz), 3.63 (bs, 2H), 3.04 (m, 1H),2.12-1.98 (bs, 1H), 1.83-1.62 (m, 2H), 1.42 (t, 2H, J=7.0 Hz), 1.16-0.95(m, 2H), 0.90 (appt, 6H, J=7.0 Hz), 0.53 (sept, 1H, J=4.3 Hz), 0.42 (dt,1H, J=8.4, 4.5 Hz), 0.34 (dt, 1H, J=8.4, 5.0 Hz); HRMS (ESI) m/z calcdfor C₁₈H₂₇NO₃Na 328.1889, found 328.1860.

TEMPO-4-yl-2-(2-((S)-1-(tert-butoxycarbonylamino)-3-methylbutyl)cyclopropyl)acetamide(14a)

To a solution of the alcohol 13a (130 mg, 0.477 mmol) in acetone (5 mL)at 0° C. was slowly added a solution of Jones reagent (2.5M, 0.48 mL,1.194 mmol). The resulting dark suspension was stirred at 0° C. for 1 h,then diluted with Et₂O and water. The aqueous phase was separated andextracted with Et₂O (2×). The combined organic layers were washed withwater (2×) and brine (1×), dried (Na₂SO₄), filtered and concentrated invacuo to yield 133 mg (97%) of the crude title compound as a colorlessoil, that was used for the next step without further purification.

To a solution of this acid (127.6 mg, 0.447 mmol, crude) in dry DCM (5.5mL) at 0° C. were added successively a solution of 4-amino-TEMPO (118.4mg, 0.671 mmol) in dry DCM (0.5 mL), DMAP (60.7 mg, 0.492 mmol),HOBt.H₂O (66.4 mg, 0.492 mmol) and EDCI (105.0 mg, 0.536 mmol). Theresulting orange solution was stirred at rt under argon for 15 h, andthen washed with sat. NH₄Cl. The aqueous phase was separated andextracted once with DCM, and the combined organic layers were dried(Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography(SiO₂, 5:5 to 3:7, hexanes/EtOAc) afforded 150.0 mg (76%) of the titlecompound as a peach colored foam, mp 139.5° C.; [α]_(D) ²³ −15.7 (c 0.5,DCM); EIMS m/z 438 ([M]⁺, 6), 252 (57), 140 (67), 124 (80), 91 (48), 84(59), 57 (100); HRMS (ED m/z calcd for C₂₄H₄₄N₃O₄ 438.3332, found438.3352.

TEMPO-4-yl-2-(2-((S)-1-(benzyoxycarbonylamino)-3-methylbutyl)cyclopropyl)acetamide(14b)

To a solution of the alcohol 13b (110.5 mg, 0.362 mmol) in acetone (5mL) at 0° C. was slowly added a solution of Jones reagent (2.5M, 0.36mL, 0.904 mmol). The resulting dark suspension was stirred at 0° C. for1 h, then diluted with Et₂O and water. The aqueous phase was separatedand extracted with Et₂O (2×). The combined organic layers were washedwith water (2×) and brine (1×), dried (Na₂SO₄), filtered andconcentrated in vacuo to yield 113.5 mg (98%) of the erode titlecompound as a colorless oil, that was used for the next step withoutfurther purification.

To a solution of this acid (110 mg, 0.344 mmol, crude) in dry DCM (4.5mL) at 0° C. were added successively a solution of 4-amino-TEMPO (91.2mg, 0.517 mmol) in dry DCM (0.5 mL), DMAP (46.7 mg, 0.379 mmol),HOBt.H₂O (51.2 mg, 0.379 mmol) and EDCI (80.8 mg, 0.413 mmol). Theresulting orange solution was stirred at rt under argon for 18 b, andthen washed with sat. NH₄Cl. The aqueous phase was separated andextracted once with DCM, and the combined organic layers were dried(Na₂SO₄), filtered and concentrated in vacuo. Flash chromatography(SiO₂, 4:6, hexanes/EtOAc) afforded 123 mg (75%) of the title compoundas a peach colored foam, mp 51.8° C. (softening point: 44° C.); [α]_(D)²³ −15.3 (c 0.5, DCM); EIMS m/z 472 ([M]⁺, 42), 415 (58), 322 (43), 168(47), 140(46), 124 (75), 91 (100), 84(53); HRMS (EI) m/z calcd forC₂₇H₄₂N₃O₄ 472.3175, found 472.3165.

Example 20—Synthesis of Alternative Nitroxide Moieties

Schematics are shown for alternative nitroxide moieties, where FIG. 42shows a synthesis protocol for5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO) and FIG.43 shows a synthesis protocol for 6-amino-1-methyl 2-azaadamantaneN-oxyl (6-amino-1-Me-AZADO).

Compounds 5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO)and (20) are shown m FIG. 42 and were prepared according to thefollowing.

Synthesis of 5-amino-TMIO was previously described by Reid, D. A. et al(The synthesis of water soluble isoindoline nitroxides and apronitroxide hydroxylamine hydrochloride UV-VIS probe for free radicals.Chem Comm. 1998, 77, 1907-8) and references cited therein.

2-Benzyl-1,1,3,3-tetramethylisoindoline (16)

(First step: Org. Synth. 1998, 9, 649; second step: Griffiths, P. G etal. Synthesis of the radical scavenger1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust J. Chem. 1983, 36,397-401). An oven-dried 250 mL, three-necked, round-bottom flask wasflushed with nitrogen, and magnesium turnings (3.84 g, 156.5 mmol) wereintroduced, that were covered with dry Et₂O (9 mL). A solution of MeI(9.45 mL, 150.2 mmol) in dry Et₂O (80 mL) was then added dropwise via adropping funnel while stirring over a period of 50 min. The resultingreaction mixture was then stirred for an additional 30 min, and thenconcentrated by slow distillation of solvent until the internaltemperature reached 80° C. The residue was allowed to cool to 60° C.,and a solution of N-benzylphthalimide (6.00 g, 25.04 mmol) in drytoluene (76 mL) was added dropwise via a dropping funnel with stirringat a sufficient rate to maintain this temperature. When the addition wascomplete, solvent was distilled slowly from the mixture until thetemperature reached 108-110° C. The reaction mixture was refluxed at110° C. for 4 h, then concentrated again by further solventdistillation. It was then cooled and diluted with hexanes (turnedpurple). The resulting slurry was filtered through Celite and washedwith hexanes. The combined yellow filtrate turned dark red-purple afterstanding in air overnight. It was then concentrated in vacuo. Theresulting purple residue was passed through a short column of basicalumina (grade I, 70-230 mesh), eluting with hexanes (˜1 L), to afford2.585 g (39%) of the title compound as a colorless oil which solidifiedto give a white solid, mp 61.0-61.4° C. ¹H NMR (300 MHz, CDCl₃) δ 7.48(appd, 2H, J=7.2 Hz), 7.34-7.19 (m, 5H), 7.18-7.11 (m, 2H), 4.00 (s,2H), 1.31 (s, 12H); HRMS (EI) m/z calcd for C₁₉H₂₃N 265.1830, found265.1824.

1,1,3,3-Tetramethylisoindoline (17)

(Griffiths, P. G. et al. Synthesis of the radical scavenger1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust. J. Chem. 1983, 36,397-401; Chan, K. S. et al. Reactions of nitroxides withmetalloporphyrin alkyls bearing beta hydrogens: aliphatic carbon-carbonbond activation by metal centered radicals. J. Organomet. Chem. 2008,693, 399407). The protected benzyl-amine 16 (1.864 g, 7.02 mmol) wasdissolved in AcOH (34 mL) in a Parr flask, and 10% Pd/C (169.5 mg) wasadded. (The reaction was splited in 3 batches.) The flask was placed ina high pressure reactor. The reactor was charged with H₂ and purged for5 cycles and was finally pressurized with H₂ at 4 bars (60 psi). Afterstirring at rt for 3 h. the reaction mixture was filtered throughCelite, and the solvent removed in vacuo. The resulting residue wasdissolved in water (5 mL) and the solution neutralized with 2.5N NaOH(pH 11.5), and extracted with Et₂O (3×50 mL). The combined organiclayers were dried (Na₂SO₄), filtered and concentrated in vacuo to yield1.165 g (95%) of the crude title compound as slightly yellow crystals,mp 36.0-36.5° C. ¹H NMR (300 MHz, CDCl₃) δ 7.30-7.23 (m, 2H), 7.18-7.11(m, 2H), 1.86 (bs, 1H), 1.48 (s, 12H).

1,1,3,3-Tetramethylisoindolin-2-yloxyl (18)

(Griffiths, P. G. et al. Synthesis of the radical scavenger1,1,3,3-tetramethylisoindolin-2-yloxyl. Aust. J. Chem. 1983, 36,397-401; Chan, K. S. et al. Reactions of nitroxides withmetalloporphyrin alkyls bearing beta hydrogens: aliphatic carbon-carbonbond activation by metal centered radicals. J. Organomet Chem. 2008,693, 399-407). To a solution of the amine 17 (1.46 g, 8.33 mmol) in a14:1 mixture of MeOH/MeCN (16.6 mL) were added successively NaHCO₃ (560mg, 6.67 mmol), Na₂WO₄.2H₂O (83.3 mg, 0.25 mmol) and 30% aq. (3.12 mL,27.50 mmol). The resulting suspension was stirred at rt. After 18 h, abright yellow suspension formed and 30% aq. H₂O₂ (3.00 mL, 26.44 mmol)was added. The reaction mixture was stirred for 2 days, then dilutedwith water and extracted with hexanes (2×). The combined organic layerswere washed with 1M H₂SO₄ and brine, dried (Na₂SO₄), filtered andconcentrated in vacuo to yield 1.55 g (98% crude) of the title compoundas a yellow crystalline powder, that was used for the next step withoutfurther purification, mp 122-125° C. (softening point: 108° C.); HRMS(EI) m/z calcd for C₁₂H₁₇NO 191.1310, found 191.1306.

5-Nitro-1,1,3,3-tetramethylisoindolin-2-yloxyl (19)

(Bolton, R. et al. An EPR and NMR study of sometetramethylisoindolin-2-yloxyl free radicals. J. Chem. Sac. Perkin Hans.2, 1993, 2049-52). Conc. H₂SO₄ (13.5 mL) was added dropwise to 18 (1.345g, 7.07 mmol) cooled in an ice-water bath, forming a dark-red solutionwhich was then warmed to 60° C. for 15 min and then cooled to 0° C.Conc. HNO₃ (0.90 mL, 19.09 mmol) was added dropwise. When the reactionappeared complete, the yellow-orange solution was heated at 100° C. for10 min, the color turning to red-orange. After cooling to rt, thereaction mixture was neutralized by careful addition to ice-cooled 2.5NNaOH (30 mL). This aqueous phase was extracted with Et₂O until it becamecolorless and the combined organic layers were dried (Na₂SO₄), filteredand concentrated in vacuo to yield 1.64 g (98%) of the crude titlecompound as a yellow-orange powder, that was used for the next stepwithout further purification.

5-amino-1,1,3,3-tetramethylisoindolin-2-yloxyl (5-amino-TMIO)

(First step: Reid, D. A. et al. The synthesis of water solubleisoindoline nitroxides and a pronitroxide hydroxylamine hydrochlorideUV-VIS probe for free radicals. Chem Comm. 1998, 77, 1907-8; Giroud, A.M. and Rassat, A. Nitroxydes LXXX: syntheses de mono et biradicauxnilroxydes dérivés de l'isoindoline. Bull. Soc. Chim. Fr. 1979, 11,48-55; second step: Keana, J. F. W. and Lee, T. D. Versatile synthesisof doxyl spin labels bypassing the usual ketone precursors. J. Am. Chem.Soc. 1975, 97, 1273-4). A flask containing a solution of 19 (1.50 g,6.38 mmol, crude) in MeOH (75 mL) was purged and filled with argon, then10% Pd/C (150 mg) was added. The flask was purged and filled 3 timeswith H₂, and the resulting black suspension was stirred at rt under H₂(1 atm) for 4 h. the reaction mixture was then filtered through Celite,the Celite washed with MeOH, and the solution concentrated in vacuo toyield 1.38 g of the crude title compound as a yellow solid, that wasused for the next step without further purification. ¹H NMR (300 MHz,CD₃OD) δ 6.89 (d, 1H, 7=8.1 Hz), 6.25 (dd, 1H, J=8.1, 2.1 Hz), 6.54 (d,1H, J=2.1 Hz), 3.35 (s, 2H), 1.34 (appd, 12H, J=5.7 Hz).

To a solution of the crude hydroxylamine (1.38 g, 6.38 mmol) in MeOH (75mL) was added Cu(OAc)₂.H₂O (26 mg, 0.128 mmol). The reaction mixture wasstirred at rt under air for 1.5 h, the color, turning to dark brown. Thesolvent was then removed in vacuo, the residue taken up in CHCl₃ and asmall amount of MeOH to dissolve the insoluble material, and washed withwater. The aqueous phase was extracted twice with CHCl₃, and thecombined organic layers were washed with brine, dried (Na₂SO₄), filteredand concentrated in vacuo. Flash chromatography (SiO₂, 6:4 to 5:5,hexanes/EtOAc) afforded 1.126 g (86%) of the title compound as a yellowpowder, mp 192-194° C. (softening point: 189° C.; HRMS (EI) m/z calcdfor C₁₂H₁₇N₂O 205.1341, found 205.1336.

TMIO-5-yl-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide (20)

To a solution of the alcohol 9a (187 mg, 0.728 mmol, prepared accordingto previous examples) in acetone (7 mL) at 0° C. was slowly added asolution of Jones reagent (2.5M, 0.73 mL, 1.821 mmol). The resultingdark suspension was stirred at 0° C. for 1 h, then diluted with Et₂O andwater. The aqueous phase was separated and extracted with Et₂O (2×). Thecombined organic layers were washed with water (2×) and brine (1×),dried (Na₂SO₄), filtered and concentrated in vacuo to yield 190 mg (96%)of the crude title compound as a slightly yellow oil, that was used forthe next step without further purification.

To a solution of this acid (187.4 mg, 0.691 mmol, crude) in dry DCM (8mL) at 0° C. were added successively 5-amino-TMIO (212.6 mg, 1.036mmol), DMAP (93.7 mg, 0.760 mmol), HOBt.H₂O (102.6 mg, 0.760 mmol) andEDCI (162.1 mg, 0.829 mmol). The resulting yellowish solution wasstirred at rt under argon for 16 h. and then washed with sat. NH₄Cl. Theaqueous phase was separated and extracted once with DCM, and thecombined organic layers were washed twice with 1 N HCl and once withsat. NaHCO₃, dried (Na₂SO₄), filtered and concentrated in vacuo. Flashchromatography (SiO₂, 6:4, hexanes/EtOAc) afforded 221.0 mg (70%) of thetitle compound as a pale orange foam, mp 78-79° C. (softening point: 70°C.); [α]_(D) ²² +72.2 (c 0.5, DCM); ESIMS m/z 481 [M+Na]⁺, 50), 939([2M+Na]⁺, 100).

Compound 6-amino-1-methyl 2-azaadamantane N-oxyl (6-amino-1-Me-AZADO)and (30) are shown in FIG. 43 and were prepared according to thefollowing.

2-Adamantanecarbonitrile (tricyclo[3.3.1.13,7]decane-2-carbonitrile, 22)

(Oldenziel, O. H. et al. 2-Adamantanecarbonitrile. Org. Synth. 1977, 57,8; Rohde. J. J. et al. Discovery and Metabolic Stabilization of Potentand Selective 2-Amino-N-(adamant-2-yl) Acetamide 11β-HydroxysteroidDehydrogenase Type 1 inhibitors. J. Med. Chem. 2007, 50, 149-64). A 3-5°C. solution of 2-adamantanone (tricyclo[3.3.1.13,7]decan-2-one, 21)(21.0 g, 137 mmol), p-tolylsulfanylmethyl isocyanide (TosMIC, 35.5 g,178 mmol) and EtOH (14 mL, 233 mmol) in 1,2-dimethoxyethane (DME, 470mL) was treated with portionwise addition of solid t-BuOK (39.2 g, 342mmol), maintaining the internal temperature below 10° C. After theaddition, the resulting slurry reaction mixture was stirred at rt for 30min and then al 35-40° C. for 30 min. The heterogeneous reaction mixturewas filtered and the solid washed with DME. The filtrate wasconcentrated in vacuo, loaded to a short Al₂O₃ column (activated,neutral, Brockmann I, 150 mesh, 7 cm thick×15 cm height), and washed offwith a 5:1 mixture of hexanes/DCM (˜1.5 L). The solution wasconcentrated in vacuo to afford 19.0 g (86%) of the title compound as awhite powder, ¹H NMR (300 MHz, CDCl₃) δ 2.91 (s, 1H), 2.23-2.08 (m, 4H),2.00-1.80 (m, 4H), 1.80-1.66 (m, 6H).

2-Adamantane carboxylic Acid (23)

(Rohde, J. J. et al. Discovery and Metabolic Stabilization of Potent andSelective 2-Amino-N-(adamant-2-yl) Acetamide 11β-HydroxysteroidDehydrogenase Type 1 Inhibitors. J. Med. Chem. 2007.50, 149-64). Amixture of the nitrile 22 (18.9 g, 117 mmol) in AcOH (56 mL) and 48% HBr(224 mL) was stirred at 120° C. overnight. The reaction mixture wascooled at 4° C., standing for 4 h, then filtered. The solid was washedwith water and dried in vacuum over silica gel overnight, to yield 20.6g (98%) of the title compound as off-white crystals, ¹H NMR (300 MHz,DMSO-4) δ 12.09 (s, 1H), 2.55-2.47 (m, 1H), 2.20 (bs, 2H), 1.87-1.64 (m,10H), 1.60-1.50 (m, 2H).

5,7-Dibromo-2-adamantane carboxylic acid (24)

(Adapted from Rohde, J. J. et al. Discovery and Metabolic Stabilizationof Potent and Selective 2-Amino-N-(adamant-2-yl) Acetamide11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors. J. Med Chan. 2007,50, 149-64). A vigorously stirred 0° C. solution of AlBr₃ (18.9 g, 69.6mmol), BBr₃ (2.40 g, 9.49 mmol) and Br₂ (40 mL) was treated portionwisewith the acid 23 (5.70 g, 31.6 mmol). Upon completion of the addition,the reaction mixture was stirred at 70° C. for 48 h, then cooled in anice bath, and quenched carefully with sat. sodium bisulfite. Stirringwas continued at rt overnight. The resultant pale brown suspension wasfiltered, the solid washed with water and dried overnight under vacuumat 60° C. to yield 10.95 g (quant.) of the crude title compound as abeige powder. ¹H NMR (300 MHz, DMSO-d₆) δ 12.56 (bs, 0.3H), 2.85 (appd,211, 7=12.9 Hz), 2.75-2.55 (m, 2H), 2.50-2.35 (m, 2H), 2.35-2.10 (m,7H).

(5,7-Dibromo-adamantan-2-yl)-carbamic acid tert-butyl ester (25)

A suspension of the acid 24 (2.00 g, 5.92 mmol) in dry toluene (30 mL)was treated successively with Et₃N (1.0 mL, 7.10 mmol) anddiphenylphosphoryl azide (DPPA, 1.6 mL, 7.10 mmol). The resultingmixture was stirred at 85° C. for 15 h. To a separated flask containinga solution of t-BuOK (1.35 g, 11.8 mmol) in dry THF (80 mL) al 0° C. wasadded the isocyanate solution dropwise via a dropping funnel. Theresulting reaction mixture was allowed to warm to rt over 30 min, andthen it was quenched with water. The THF was removed in vacuo, and theresulting material was diluted with EtOAc. The organic layer was washedwith 1 N HCl, sat. NaHCO₃ and brine, dried (Na₂SO₄), filtered andconcentrated in vacuo. Flash chromatography (SiO₂, 95:5 to 8:2,hexanes/EtOAc) afforded 1.20 g (50%, 2 steps) of the title compound as awhite powder. ¹H NMR (300 MHz, CDCl₃) δ 4.68 (bs, 1H), 3.76 (bs, 1H),2.87 (s, 2H), 2.47-2.13 (m, 10H), 1.46 (s, 9H).

(7-Methylene-bicyclo[3.3.1]nonan-3-one-9-yl)-carbamic acid tert-butylester (26)

(First step: Rohde, J. J. et al. Discovery and Metabolic Stabilizationof Potent and Selective 2-Amino-N-(adamant-2-yl) Acetamide11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitors. J. Med. Chem. 2007,50, 149-64). A solution of 25 (125 mg, 0.305 mmol) in dioxane (0.80 mL)was treated with 2N NaOH (0.70 mL, 1.37 mmol) and irradiated undermicrowaves (μw, Biotage) for 15 min at 180° C. The dioxane was removedin vacuo. The residue was dissolved in DCM, washed with water, dried(Na₂SO₄), filtered and concentrated in vacuo to afford 82.5 mg of crude9-amino-7-methylene-bicyclo[3.3.1]nonan-3-one as a yellow oil, that wasused for the next step without further purification.

To a solution of this crude amine in dry DCM (5 mL) was added Et₃N (0.13mL, 0.913 mmol) and then Boc₂O (73.8 mg, 0.335 mmol) at 0° C. Thereaction mixture was stirred at rt under N₂ for 14 h. The reaction wasquenched with sat. aq. NH₄Cl and the aqueous phase extracted twice withDCM. The combined organic layers were dried (Na₂SO₄), filtered andconcentrated in vacuo. Flash chromatography (SiO₂, 7:3, hexanes/EtOAc)afforded 48.0 mg (59%, 2 steps) of the title compound as a white powder.¹H NMR (300 MHz, CDCl₃) δ 4.93 (bs, 0.25H), 4.84 (s, 2H), 4.81 (bs,0.75H), 4.12 (bs, 0.25H), 3.91 (appbd, 0.75H, J=3.6 Hz), 2.64-2.37 (m,6H), 2.37-2.23 (m, 3.25H), 2.17 (appbd, 0.75H, J=13.8 Hz), 1-48 and 1.46(2 s, 9H).

(7-Methylene-bicyclo[33.1]nonan-3-one oxime-9-yl)-carbamic acidtert-butyl ester (27)

To a solution of ketone 26 (137 mg, 0.515 mmol) in dry pyridine (1 mL)was added NH₂OH.HCl (109 mg, 1.54 mmol). The reaction mixture wasstirred at rt under argon for 23 h. The solvent was then removed invacuo, and the residue was diluted with EtOAc and diet) water was added.The layers were separated and the aqueous phase extracted with EtOAc.The combined organic layers were washed with 5% aq. CuSO₄ (3×), brine(1×), dried (Na₂SO₄), filtered and concentrated in vacuo. Flashchromatography (SiO₂, 4:6, hexanes/EtOAc) afforded 133 mg (92%) of thetitle compound as a colorless gum. ¹H NMR (300 MHz, CDCl₃) δ 7.02 (bs,0.6H), 4.90 (bs, 0.25H), 4.80 (d, 1H, 7=2.1 Hz), 4.76 (bs, 0.75H), 4.69(d, 1H, J=2.1 Hz), 3.87 (bs, 1H), 3.26 (d, 0.25H, J=16.8 Hz), 3.11 (d,0.75H, J=16.8 Hz), 2.55-2.48 (m, 4H), 2.48-2.20 (m, 4H). 2.16 (appd,0.25H, J=17.1 Hz), 2.04 (dd, 0.75H, J=17.1, 5.4 Hz), 1.47 (s, 9H).

(1-Iodomethyl-2-azaadamantan-6-yl)-carbamic acid tert-butyl ester (28)

To a mixture of oxime 27 (130 mg, 0.464 mmol) and MoO₃ (94 mg, 0.649mmol) in dry MeOH (4.6 mL) at 0° C. under argon was added NaBH₄ (179 mg,4.64 mmol) portion wise. The reaction mixture was stirred at 0° C., and2 additional amounts of NaBH₄ (179 mg, 4.64 mmol) were added portionwiseafter 2.5 h and after 5.5 h. After 7 h, the dark brown reaction mixturewas quenched with acetone and then filtered through Celite, and theCelite rinsed with acetone. The filtrate was concentrated in vacuo. Theresulting residue was diluted with water and extracted twice with EtOAc.The combined organic layers were washed with brine, dried (K₂CO₃),filtered and concentrated in vacuo to afford 136 mg of the crude amineas a yellow oil, that was used for the next step without furtherpurification.

To a suspension of this crude amine in dry acetonitrile (MeCN, 2.3 mL)at 0° C. under argon was added I₂ (117 mg, 0.462 mmol). The reactionmixture was allowed to stir at rt for 4 h and then quenched with sat.NaHCO₃ and sat. Na₂S₂O₃. The resulting mixture was extracted twice withDCM/CHCl₃, and the organic layer was dried (K₂CO₃), filtered andconcentrated in vacuo. Mash chromatography (SiO₂, 95:5 to 9:1, DCM/MeOH)afforded 76.5 mg (42%) of the title compound as a brown oil. ¹H NMR (300MHz, CDCl₃) δ 4.83 (bs, 1H), 3.77 (bs, 1H), 3.30 (bs. 1H), 3.24 (apps,2H), 2.14 (appbs, 2H), 1.94 (appbd, 2H, J=13.5 Hz), 1.75 (m, 6H), 1.46(s, 9H).

(1-Methyl-2-azaadamantane-N-oxyl-6-yl)-carbamic acid tert-butyl ester(29)

Deiodination of the amine 28 can be achieved by treating 28 with areducing agent, such as LiAlH₄ or NaBH₄, possibly in the presence of acatalyst, such as InCl₃, and in a polar aprotic solvent such as THF orMeCN.

Oxidation of the resulting amine to afford the corresponding nitroxide29 can be achieved by treating the said amine with H₂O₂ in the presenceof a catalytic amount of Na₂WO₄.2H₂O, in a solvent mixture of MeOH andH₂O.

6-Amino-1-methyl-2-azaadamantane-N-oxyl (6-amino-1-Me-AZADO)

Cleavage of the Boc-protecting group can be achieved by treating theprotected amine 29 with trifluoroacetic acid (TFA) in DCM, to afford thefree amine 6-amino-1-Me-AZADO.

(1-Me-AZADO-6-yl)-(S,E)-5-(tert-butoxycarbonylamino)-7-methyloct-3-enamide(30)

Jones oxidation of (S,E)-tert-butyl8-hydroxy-2-methyloct-5-en-4-ylcarbamate (9a) affords the correspondingacid as described above. Compound (9a) is prepared according to previousexamples.

Amide coupling of the said acid with 6-amino-1-Me-AZADO is achievedfollowing the conditions described above, using the coupling agentsEDCI, DMAP, and HOBt-hydrate in CH₂Cl₂ (DCM), to yield compound (30).

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those skilledin the art that numerous variations of the details of the presentinvention may be made without departing from the invention as defined inthe appended claims.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within theslated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values.

1-23. (canceled)
 24. A compound having the structure

wherein X is one of

and R₁ is hydrogen, C₁-C₆ straight or branched-chain alkyl, or a C₁-C₆straight or branched-chain alkyl further comprising a phenyl (C₆H₅)group, that is unsubstituted or is methyl-, hydroxyl- orfluoro-substituted; R₄ is hydrogen, C₁-C₆ straight or branched-chainalkyl, or a C₁-C₆ straight or branched-chain alkyl further comprising aphenyl (C₆H₅) group, that is unsubstituted or is methyl-, hydroxyl- orfluoro-substituted; R₃ is —NH—R₅, —O—R₅ or —CH₂—R₅, and R₅ is an —N—O,—N—OH or N═O containing group; and R is —C(O)—R₆, —C(O)O—R₆, or—P(O)—(R₆)₂, wherein R₆ is C₁-C₆ straight or branched-chain alkyl orC₁-C₆ straight or branched-chain alkyl further comprising one or morephenyl (—C₆H₅) groups that are independently unsubstituted, or methyl-,ethyl-, hydroxyl- or fluoro-substituted.
 25. The compound of claim 24having the structure


26. The compound of claim 24 having the structure

wherein R₄ is hydrogen or methyl. 27-39. (canceled)
 40. The compound ofclaim 24, wherein R₅ has the structure


41. The compound of claim 24, wherein R is Ac, Hoc, Cbz, or —P(O)-Ph₂.42. The compound of claim 24, in which R₁, R₄ and R₆ are independentlychosen from hydrogen, methyl, ethyl, propyl, 2-propyl, butyl, t-butyl,pentyl, hexyl, benzyl, and hydroxybenzyl.
 43. The compound of claim 24wherein X is


44. The compound of claim 24, wherein X is


45. The compound of claim 24, wherein R₅ has the structure:


46. The compound of claim 25, wherein R₅ has the structure:


47. The compound of claim 26, wherein R₅ has the structure:


48. The compound of claim 42, wherein R₅ has the structure:


49. A compound of structure:


50. A compound of structure: